Reagents and methods useful for detecting diseases of the urinary tract

Polypeptides and polynucleotides useful for detecting, diagnosing, staging, monitoring, prognosticating, in vivo imaging, preventing or treating, or determining the predisposition of an individual to diseases and conditions of the urinary tract, such as urinary cancer, are described. These sequences are derived from keratin/cytokeratin, CAS, or mat-8 polypeptides and polynucleotides. Also provided are antibodies which specifically bind to keratin/cytokeratin, CAS, or mat-8-encoded polypeptides or proteins, which molecules are useful for the therapeutic treatment of urinary tract diseases, tumors or metastases.

BACKGROUND OF THE INVENTION
 The invention relates generally to detecting diseases of the urinary tract.
 Furthermore, the invention also relates to reagents and methods for
 detecting diseases of the urinary tract. More particularly, the present
 invention relates to reagents such as polynucleotide sequences and the
 polypeptide sequences encoded thereby, as well as methods which utilize
 these sequences. The polynucleotide and polypeptide sequences are useful
 for detecting, diagnosing, staging, monitoring, prognosticating, in vivo
 imaging, preventing or treating, or determining predisposition to diseases
 or conditions of the urinary tract such as urinary tract cancers.
 The organs of the urinary tract include the bladder, kidneys, and ureter.
 The incidence of urinary tract cancers in the United States is projected
 to be 86,300 cases diagnosed and 24,700 related deaths to occur during
 1998. The most prevalent of the urinary tract cancers is bladder cancer,
 with projections of 54,400 new cases diagnosed and 12,500 related deaths
 to occur during 1998 (American Cancer Society statistics). Bladder tumors
 are heterogeneous in their ability to progress and are characterized by a
 high rate of recurrence. Hence, bladder cancer patients are monitored
 closely following their initial treatment.
 Diseases such as bladder cancer traditionally have been diagnosed by
 appearance of blood in urine (hematuria) and confirmed by more detailed
 visualization of cells in biopsy samples under a microscope by highly
 trained personnel. The standard surveillance technique to detect recurrent
 bladder cancer is cystoscopy. Flexible cystoscopes have made cystoscopy
 more acceptable to patients but the method remains invasive. Efforts to
 replace cystoscopy by examining voided urine for tumor cells have included
 cytopathology and flow cytometry but the sensitivities are not high enough
 to detect the majority of recurrence, particularly those that are well or
 moderately differentiated. Cost also limits their use as adjuncts to
 cystoscopy. Other urinary markers that might be useful for diagnosing
 recurrences are being developed, including nonspecific markers, such as
 various growth factors, immune complexes and tumor-related proteins.
 However, to date, no single test has proved reliable enough to gain
 widespread acceptance. In particular, no test to date has sufficient
 simplicity, sensitivity, specificity and cost effectiveness to warrant
 pre-symptomatic screening.
 Following is a more detailed summary of methods and markers in urine that
 have been used for detection of bladder tumors, and their limitations:
 Hematuria is found in up to 20% of a target population (Mariani A J,
 Mariani M C, Macchioni C, Stams U K, Hariharan A, Moriera A. J Urol
 141:350-355, 1989; Messing E M, Young T B, Hunt V B, Roecker E B,
 Vailiancourt A M, Hisgen W J, Greenberg E B, Kuglitsch M E, Wegenke, J D:
 J Urol 148:289-292, 1992; and Britton J P, Dowell A C, Whelan P, Harris C
 M. J Urol 148:788-790, 1992), and 4-9% of those will have malignancy. In
 one of the most recent studies, the BTA (basement membrane complexes) test
 showed sensitivity of 40%, 96% specificity for healthy volunteers and
 80-90% specificity for patients with non-malignant genitourinary disease
 (Sarosdy M F, deVere White R W, Soloway M S, Sheinfeld J, Hudson M A,
 Schelihammer P F, Jarowenko M V, Adams G, Blumenstein B A: J Urol
 154:379-384, 1995). Urinary basement membrane antigens have been found
 higher in invasive cancer, with sensitivity 58% and specificity 96% (Abou
 Farha K M M, Janknegt R A, Kester A D M, Arendt J W: Urol Int 50 133-140,
 1993). The Aura-Tek FDP dipstick showed 69% sensitivity with a patient
 population and 96% specificity with a healthy population (Schmetter B S,
 Habicht K K, Lamm D L, Morales A, Grossman H B, Bander N, Hanna M G,
 Butman BR : J Urol 155:492A, 1996). Nuclear matrix protein NMP22 showed
 considerable overlap between patients with tumors and benign urological
 conditions (Carpinita G A, Stadler W M, Briggman J V, Chodak G W, Church P
 A, Lamm D L, Lange P H, Messing E M, Pasciak R M, Reservitz G B, Ross R N,
 Rukstalis D B, Sarosday M F, Soloway M S, Thiel R P, Vogelzang N, Hayden C
 L: J Urol 156:1280-1285, 1996). Carcinoembryonic antigen (CEA) has been
 shown to occur in urine of some patients with bladder cancer, but the
 potential diagnostic utility is unclear (Wahren B, Edsmyr F: Urol Res
 6:221-224, 1978). High levels of autocrine motility factor (AMF) have been
 detected in urine of patients with bladder cancer, showing correlation
 with stage and grade, but the clinical utility has not been established
 (Guirguis R, Schiffman E, Liu B, Birbeck D, Engel J, Liotta L: J Nat
 Cancer Inst 80:1203-1211, 1988). Similarly, high levels of fibroblast
 growth factors (FGF) have been found in urine of some bladder cancer
 patients (O'Brien T S, Smith K, Cranston D, Fuggle S, Bicknell R, Harods A
 L: Brit J Urol 76:311-314, 1995; Nguyen M, Watanabe H, Budson E, Richie J
 P, Folkman J: J Nat Cancer Inst 85:241-242, 1993; Chopin D K, Caruelle
 J-P, Colmbel M, Pallcy S, Ravery V, Caruelle D, Abbout C C, Bardtault D: J
 Urol 160:1126-1130, 1991). Further, high urinary levels of Scatter
 Factor/Hepatocyte Growth Factor (SF/HGF) have been reported in patients
 with bladder cancer, but the data is as yet unconfirmed (Joseph A, Weiss G
 H, Lin L, Fuchs A, Chowdhury S, O'Shaugnessy P, Goldbert I D, Rosen E M: J
 Nat Cancer Inst 87:372-377, 1995). In one unconfirmed report, urinary type
 IV collagenase has been found to be higher in urine from patients with
 invasive cancers (Margulies I M K, Hoyhtya M, Evans C, Stracke M L, Liotaa
 L A, Stetler-Stevenson W G: Cancer Epidemiol Biomarkers Prev 1:467-474,
 1992). Hyaluronic acid has recently been proposed as a 93% specific and
 92% sensitive marker (Lokeshwar V B, Obeck C, Soloway M S, Block N L:
 Cancer res 57, 774-777, 1997), but the data remains to be confirmed by
 larger studies. Patent application EP 0678744A2 describes a urinary tumor
 associated protein which is characterized by being immunogenic in cancer
 patients. The antigen was not characterized in detail, and is unlikely to
 be a member of the keratin/cytokeratin family, since these are not known
 to be immunogenic in cancer patients. There is thus a great effort to
 discover specific urinary markers, but none have become routinely
 established.
 Assays for urinary tumor markers based on proteins of the
 keratin/cytokeratin family have been described. Some of these assays use
 uncharacterized antibodies while others use antibodies with defined
 specificity. As suggested below, the basis of these assays appears to be
 selective release of soluble peptide fragments by tumorous cells.
 Furthermore, the literature suggests that keratins and cytokeratins are
 characteristic of the differentiated state of cells, and, where present,
 are indicative of the normal cellular origin of the tumor, not of the
 state of de-differentiation. Indeed, Nagle [Cancer & Metastasis Rev.
 15:473-482 (1996)] states that intermediate filaments in general,
 including the cytokeratins as a class, are valuable markers for
 distinguishing the cellular origin of various undifferentiated neoplasms.
 Sundstrom and Stigbrand (International Journal of Biological markers, 9,
 102-108, 1994) state that cytokeratin markers are characteristic of the
 expression patterns of normal epithelial cells and the retention of the
 pattern is a useful means for classifying tumors. These authors also
 describe Tissue Polypeptide Antigen (TPA) as a complex of cytokeratins 8,
 18 and 19. Carbin, Eckman and Eneroth (Urol Res 17, 269-272, 1989) state
 that TPA in serum and urine is the result of increased turnover and
 autolysis of malignant cells. Because of the poly-specificity of the TPA
 assays, any selectivity due to de novo synthesis would not be apparent,
 and, should only be detectable with specificity using probes or antibodies
 to epitopes which are unique for the specific keratin/cytokeratins which
 are up-regulated in the cancerous tissue. U.S. Pat. No. 533,832 describes
 a monoclonal antibody with reactivity to cytokeratins 8, 18 and 19, which
 they suggest might be useful for diagnosing cancer. There is no discussion
 of up-regulated proteins indicative of bladder cancer.
 Attempts have been made to improve the specificity of assays by using TPA
 in combination with other markers (Halim A B, El-Amahdy O, Hamza S,
 Aboul-Ela M, Oehr P: International Journal of Biological markers, 7,
 234-239, 1992; Casetta G, Piana P, Cavallini A, Vottero M, Tizzani A: Brit
 J Urol 72, 60-64, 1993). The specificity is still inadequate since false
 positives due to urinary tract infections may occur. Further, a diagnostic
 test based on a single marker is preferable to an assay using three
 markers. There have also been attempts to improve the TPA assay by the use
 of monoclonal antibodies (Sundstrom B E, d'Amico Y, Brundell J: Int J Biol
 Markers, 10, 166-173, 1995). However, these monoclonal antibodies were
 used only in an attempto mimic the oligo-specificity of the polyclonal
 antibody assay, and not to improve the specificity by looking at specific
 peptides up-regulated in the cancerous tissue.
 Keratin 18 has been proposed for use as a target in more specific assays
 (Baker W C, White R D, Rossito P V, Min B H, Cardiff R D: J Urol 140,
 436-439, 1988). However, the monoclonal antibodies used had
 cross-reactivity with keratin 8 (Rossito P V, Chan R, Strand M A, Miller C
 H, Baker W C, Deitsch A D, Devere White R, Cardiff R D: J Urol 140,
 431-435, 1988). Furthermore, limited sensitivity and specificity was
 noted. Patent application PCT 95/31728 describes specific epitopes
 characteristic of cytokeratin 18 and detectable amounts of these epitopes
 in the serum of bladder cancer patients. However, the utility using these
 specific epitopes may be limited because of the specificity of the
 epitopes described. Further there is no suggestion of a general approach
 to look for keratins/cytokeratins up-regulated in bladder tumors. A
 commercial kit is available for cytokeratin 19, under the name Cyfra 21-1.
 With that kit, problems occur due to the occurrence of cells and cell
 debris in urine which must be first removed in order to determine soluble
 fragments (Dittado R, Bariolli P, Gion M, Mione R, Barichello M, Capitanio
 G, Cocco G, Cazzolato G, de Biasi F, Praturlon F, Antinozzi R, Gianneo E:
 Clin Chem 42, 1634-1638, 1996). False positives were obtained in patients
 with cystitis (Senga Y, Kimura G, Hattori T, Yoshida K: Urology 48,
 703-710, 1996). None of the above works determined the merits of markers
 which are specifically up-regulated in bladder tumors. In a comprehensive
 study, Moll et al (Moll R, Achtstatter T, Balcarova-Stander J, Ittensohn M
 and Franke WW: Amer J Pathol 132, 123-144, 1988) showed that cytokeratins
 7, 8, 13, 19 are present in normal urothelium, and that 13 was greatly
 reduced in grade three transitional cell carcinoma. No mention was made of
 any species being present in increased amounts in the cancer.
 Brinkman et al (Proc Natl Acad Sci USA 1995 October 24;92(22): 10427-10431)
 have discovered a gene referred to as CAS (cellular apoptosis
 susceptibility). They have also observed that this gene is amplified and
 has increased expression in certain tumorous cell lines from leukemia,
 colon and breast cancer (Genome Res 1996 March; 6(3): 187-194). See, also,
 PCT Publication No. WO 9640713.
 Morrison et al (Oncogene 1994, vol 9 pp 3417-3426, Journal of Biological
 Chemistry, vol 270, pp 2176-2182) have described a class of proteins which
 are expressed in tumors initiated by the oncogene neu but absent from
 those initiated by c-myc. One of these, a chloride channel protein, mat-8,
 was proposed as having diagnostic utility for breast cancer (PCT
 application WO96/05322).
 However, to date, there has been no suggestion for using the above proteins
 as diagnostic markers for urinary tract diseases.
 SUMMARY OF THE INVENTION
 The present invention is based on the surprising finding of
 keratin/cytokeratin and other markers up-regulated in urinary cancerous
 conditions but not expressed in the normal bladder. Thus, the present
 invention provides a method of detecting the presence of urinary tract
 disease in an individual which comprises providing a test sample from the
 individual and contacting the test sample with at least one
 keratin/cytokeratin, CAS, or mat-8-specific polynucleotide or complement
 thereof, wherein the keratin/cytokeratin, CAS, or mat-8-specific
 polynucleotide has at least 50% identity with a polynucleotide selected
 from the group consisting of SEQUENCE ID NOS 1 through 13, and fragments
 or complements thereof; and detecting the presence of target
 keratin/cytokeratin, CAS, or mat-8 polynucleotides in the test sample
 which bind to the keratin/cytokeratin, CAS, or mat-8-specific
 polynucleotide, as an indication of urinary tract disease in the
 individual. The keratin/cytokeratin, CAS, or mat-8-specific polynucleotide
 may be attached to a solid phase prior to performing the method.
 Furthermore, in certain embodiments, the urinary tract disease is cancer.
 The present invention also provides a method of detecting the presence of
 urinary tract disease in an individual which comprises (a) providing a
 test sample from the individual and performing reverse transcription on
 said sample using at least one primer in order to produce cDNA; (b)
 amplifying the cDNA obtained from step (a) using keratin/cytokeratin, CAS,
 or mat-8 oligonucleotides as sense and antisense primers to obtain
 keratin/cytokeratin, CAS, or mat-8 amplicon; and (c) detecting the
 presence of the keratin/cytokeratin, CAS, or mat-8 amplicon as an
 indication of urinary tract disease in the individual, wherein the
 keratin/cytokeratin, CAS, or mat-8 oligonucleotides utilized in steps (a)
 and (b) have at least 50% identity with a sequence selected from the group
 consisting of SEQUENCE ID NOS 1 through 13, and fragments or complements
 thereof. This reaction can be a direct or an indirect reaction. Further,
 the detection step can comprise utilizing a detectable label capable of
 generating a measurable signal. The detectable label can be attached to a
 solid phase. The test sample can also be reacted with a solid phase prior
 to performing one or more of the steps of the method. Further, the
 detection step may utilize a detectable label capable of generating a
 measurable signal. Additionally, in certain embodiments, the urinary tract
 disease is cancer.
 The invention further provides a method of detecting the presence of
 urinary tract disease in an individual which comprises (a) providing a
 test sample from the individual and contacting the test sample with at
 least one keratin/cytokeratin, CAS, or mat-8 oligonucleotide as a sense
 primer and with at least one keratin/cytokeratin, CAS, or mat-8
 oligonucleotide as an anti-sense primer and amplifying to obtain a first
 stage reaction product; (b) contacting the first stage reaction product
 with at least one other keratin/cytokeratin, CAS, or mat-8 oligonucleotide
 to obtain a second stage reaction product, with the proviso that the other
 keratin/cytokeratin, CAS, or mat-8 oligonucleotide is located 3' to the
 keratin/cytokeratin, CAS, or mat-8 oligonucleotides utilized in step (a)
 and is complementary to said first stage reaction product; and (c)
 detecting the second stage reaction product as an indication of urinary
 tract disease in the individual, wherein the keratin/cytokeratin, CAS, or
 mat-8 oligonucleotides utilized in steps (a) and (b) have at least 50%
 identity with a sequence selected from the group consisting of SEQUENCE ID
 NOS 1 through 13, and fragments or complements thereof. The test sample
 can be reacted with a solid phase prior to performing one or more of the
 steps of the method. Further, the detection step may utilize a detectable
 label capable of generating a measurable signal. In certain embodiments,
 the urinary tract disease is cancer.
 The invention also provides a test kit useful for detecting urinary tract
 disease in a test sample. The test kit comprises a container containing at
 least one keratin/cytokeratin, CAS, or mat-8 polynucleotide having at
 least 50% identity with a sequence selected from the group consisting
 SEQUENCE ID NOS 1 through 13, and fragments or complements thereof. Also
 provided is a test kit useful for detecting urinary tract cancer in a test
 sample which comprises a container containing a keratin/cytokeratin, CAS,
 or mat-8 polypeptide encoded by a nucleic acid sequence having at least
 50% identity with a sequence selected from the group consisting of
 SEQUENCE ID NOS 1 through 13, and fragments or complements thereof. The
 polypeptides of the test kit may be attached to a solid phase. These test
 kits further comprise containers with tools useful for collecting test
 samples (such as, for example, blood, urine, saliva and stool). Such tools
 include lancets and absorbent paper or cloth for collecting and
 stabilizing blood; swabs for collecting and stabilizing saliva; and cups
 for collecting and stabilizing urine or stool samples. Collection
 materials, such as, papers, cloths, swabs, cups, and the like, may
 optionally be treated to avoid denaturation or irreversible adsorption of
 the sample. The collection materials also may be treated with or contain
 preservatives, stabilizers or antimicrobial agents to help maintain the
 integrity of the specimens.
 Also provided is a specific binding molecule which specifically binds to a
 keratin/cytokeratin, CAS, or mat-8 epitope. The keratin/cytokeratin, CAS,
 or mat-8 epitope is derived from a polypeptide encoded by a nucleic acid
 sequence having at least 50% identity with a sequence selected from the
 group consisting of SEQUENCE ID NOS 1 through 13, and fragments or
 complements thereof. The specific binding molecule may be an antibody
 molecule of fragment thereof.
 Test kits useful for detecting urinary tract cancer in a test sample are
 also provided which comprise a container containing a specific binding
 molecule which specifically binds to a keratin/cytokeratin, CAS, or mat-8
 antigen having a keratin/cytokeratin, CAS, or mat-8 epitope. The specific
 binding molecule may be an antibody or fragment thereof and may be
 attached to a solid phase. These test kits further comprise containers
 with tools useful for collecting test samples (such as, for example,
 blood, urine, saliva and stool). Such tools include lancets and absorbent
 paper or cloth for collecting and stabilizing blood; swabs for collecting
 and stabilizing saliva; and cups for collecting and stabilizing urine or
 stool samples.
 Collection materials, such as, papers, cloths, swabs, cups, and the like,
 may optionally be treated to avoid denaturation or irreversible adsorption
 of the sample.
 The collection materials also may be treated with or contain preservatives,
 stabilizers or antimicrobial agents to help maintain the integrity of the
 specimens.
 Also provided is a method of detecting the presence of urinary tract cancer
 in an individual which comprises (a) providing a test sample from the
 individual and contacting the test sample with a specific binding molecule
 which specifically binds to an epitope of a keratin/cytokeratin, CAS, or
 mat-8 antigen selected from the group consisting of a polypeptide encoded
 by a nucleic acid sequence having at least 50% identity with a sequence
 selected from the group consisting of SEQUENCE ID NOS 1 through 13, and
 fragments or complements thereof. The contacting is performed for a time
 and under conditions sufficient for the formation of binding
 molecule/antigen complexes. Detection of the complexes is an indication of
 urinary tract cancer in the individual. The specific binding molecule may
 be an antibody molecule or a fragment thereof and may be attached to a
 solid phase.
 The invention further provides a method of detecting the presence of
 urinary tract cancer in an individual which comprises (a) providing a test
 sample from the individual and contacting the test sample with a
 keratin/cytokeratin, CAS, or mat-8 polypeptide. The keratin/cytokeratin,
 CAS, or mat-8 polypeptide contains at least one keratin/cytokeratin, CAS,
 or mat-8 epitope of a polypeptide encoded by a nucleic acid molecule
 having at least 50% identity with a sequence selected from the group
 consisting of SEQUENCE ID NOS 1 through 13, and fragments or complements
 thereof. The contacting is performed for a time and under conditions
 sufficient to allow antigen/antibody complexes to form. Detection of the
 complexes is an indication of urinary tract cancer in the individual. The
 keratin/cytokeratin, CAS, or mat-8 polypeptide can be attached to a solid
 phase.
 Also provided is a method for producing antibodies which specifically bind
 to keratin/cytokeratin, CAS, or mat-8 antigen, comprising administering to
 an individual an isolated immunogenic polypeptide or fragment thereof in
 an amount sufficient to elicit an immune response. The immunogenic
 polypeptide comprises at least one keratin/cytokeratin, CAS, or mat-8
 epitope derived from a polypeptide encoded by a nucleic acid molecule
 having at least 50% identity with a sequence selected from the group
 consisting of SEQUENCE ID NOS I through 13, and fragments or complements
 thereof.
 An additional method for producing antibodies which specifically bind to a
 keratin/cytokeratin, CAS, or mat-8 antigen is provided which comprises
 administering to an individual a plasmid comprising a sequence which
 encodes at least one keratin/cytokeratin, CAS, or mat-8 epitope derived
 from a polypeptide encoded by a nucleic acid molecule having at least 50%
 identity with a sequence selected from the group consisting of SEQUENCE ID
 NOS lthrough 13, and fragments or complements thereof.
 In another embodiment of the invention, specific binding molecules, such as
 antibodies or fragments thereof against the keratin/cytokeratin, CAS, or
 mat-8 antigens, can be used to detect antigen, or for image localization
 of the antigen, in a patient for the purpose of detecting or diagnosing a
 disease or condition. Such antibodies can be polyclonal or monoclonal, or
 made by molecular biology techniques, and can be labeled with a variety of
 detectable labels, including but not limited to radioisotopes and
 paramagnetic metals. Furthermore, antibodies or fragments thereof, whether
 monoclonal, polyclonal, or made by molecular biology techniques, can be
 used as therapeutic agents for the treatment of diseases characterized by
 expression of the keratin/cytokeratin, CAS, or mat-8 antigen. In the case
 of therapeutic applications, the antibody may be used without
 derivitization, or it may be derivitized with a cytotoxic agent such as a
 radioisotope, enzyme, toxin, drug, prodrug, or the like.

DETAILED DESCRIPTION OF THE INVENTION
 A general strategy for discovering genes and gene products which are
 specifically up-regulated in urinary tract cancers, is provided. A method
 for detecting a urinary tract cancer antigen in a test sample from an
 individual suspected of having a urinary tract cancer is provided, which
 comprises contacting the test sample with an antibody or fragment thereof
 which specifically binds to at least one urinary tract cancer antigen, for
 a time and under conditions sufficient for the formation of
 antibody/antigen complexes, and detecting the complex containing the
 antibody. The antibody can be attached to a solid phase and be either a
 monoclonal or polyclonal antibody. The antibody specifically binds to a
 polypeptide epitope characterized by being up-regulated in urinary tract
 cancers as detailed below. These polypeptides include a
 keratin/cytokeratin, CAS or mat-8 polypeptide, or fragments thereof.
 A method for producing antibodies to urinary tract cancer gene products
 comprising administering to an individual an isolated immunogenic
 polypeptide or fragment thereof comprising at least one urinary tract
 cancer epitope in an amount sufficient to produce an immune response, also
 is provided.
 The present invention provides methods for assaying a test sample for
 products of a gene characterized by being up-regulated in urinary tract
 cancers.
 Test samples which may be assayed by the methods provided herein include
 tissues, cells, body fluids and secretions. The present invention also
 provides reagents such as oligonucleotide primers and polypeptides which
 are useful in performing these methods.
 In addition to urinary tract cancer, the markers represented by SEQUENCE ID
 NOS 1-13, and polypeptides encoded thereby, may also be up-regulated in
 other urinary tract diseases or conditions of the urinary tract including,
 but not limited to, cystitis, interstitial cystitis, urethritis,
 nephrosclerosis, and nephritis.
 Portions of the nucleic acid sequences disclosed herein are useful as
 primers for the reverse transcription of RNA or for the amplification of
 cDNA; or as probes to determine the presence of certain mRNA sequences in
 test samples.
 Also disclosed are nucleic acid sequences which permit the production of
 encoded polypeptide sequences which are useful as standards or reagents in
 diagnostic immunoassays, as targets for pharmaceutical screening assays
 and/or as components or as target sites for various therapies. Monoclonal
 and polyclonal antibodies directed against at least one epitope encoded by
 these polynucleotide sequences are useful as delivery agents for
 therapeutic agents as well as for diagnostic tests and for screening for
 diseases or conditions associated with urinary tract cancer. Isolation of
 sequences of other portions of the genes of interest can be accomplished
 utilizing probes or PCR primers derived from these nucleic acid sequences.
 This allows additional probes of the mRNA or cDNA of interest to be
 established, as well as the corresponding encoded polypeptide sequences.
 These additional molecules are useful in detecting, diagnosing, staging,
 monitoring, prognosticating, in vivo imaging, preventing or treating, or
 determining the predisposition to diseases and conditions of the urinary
 tract, such as urinary tract cancer.
 Techniques for determining amino acid sequence similarity are well-known in
 the art. In general, "similarity" means the exact amino acid to amino acid
 comparison of two or more polypeptides at the appropriate place, where
 amino acids are identical or possess similar chemical and/or physical
 properties such as charge or hydrophobicity. A so-termed "percent
 similarity" then can be determined between the compared polypeptide
 sequences. Techniques for determining nucleic acid and amino acid sequence
 identity also are well known in the art and include determining the
 nucleotide sequence of the mRNA for that gene (usually via a cDNA
 intermediate) and determining the amino acid sequence encoded thereby, and
 comparing this to a second amino acid sequence. In general, "identity"
 refers to an exact nucleotide to nucleotide or amino acid to amino acid
 correspondence of two polynucleotides or polypeptide sequences,
 respectively.
 Two or more polynucleotide sequences can be compared by determining their
 "percent identity." Two or more amino acid sequences likewise can be
 compared by determining their "percent identity." The percent identity of
 two sequences, whether nucleic acid or peptide sequences, is the number of
 exact matches between two aligned sequences divided by the length of the
 shorter sequences and multiplied by 100. An approximate alignment for
 nucleic acid sequences is provided by the local homology algorithm of
 Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This
 algorithm can be extended to use with peptide sequences using the scoring
 matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M.
 O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research
 Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids
 Res. 14(6):6745-6763 (1986). An implementation of this algorithm for
 nucleic acid and peptide sequences is provided by the Genetics Computer
 Group (Madison, Wis.) in their BestFit utility application. The default
 parameters for this method are described in the Wisconsin Sequence
 Analysis Package, Program Manual, Version 8 (1995) (available from
 Genetics Computer Group, Madison, Wis.). Other equally suitable programs
 for calculating the percent identity or similarity between sequences are
 generally known in the art.
 The compositions and methods described herein will enable the
 identification of certain markers as indicative of urinary tract cancer;
 the information obtained therefrom will aid in the detecting, diagnosing,
 staging, monitoring, prognosticating, in vivo imaging, preventing or
 treating, or determining diseases or conditions associated with SEQUENCE
 ID NOS 1 through 13, and polypeptides encoded thereby, which have been
 found to be upregulated in urinary tract cancer. Test methods include, for
 example, probe assays which utilize the sequence(s) provided herein and
 which also may utilize nucleic acid amplification methods such as the
 polymerase chain reaction (PCR), the ligase chain reaction (LCR), and
 hybridization.
 In addition, the nucleotide sequences provided herein contain open reading
 frames from which an immunogenic epitope may be found. This epitope is
 believed to be unique to urinary tract cancer associated with markers
 represented by SEQUENCE ID NOS 1 through 13. It also is thought that the
 polynucleotides or polypeptides and protein encoded by SEQUENCE ID NOS I
 through 13 are useful as markers. These markers are elevated in urinary
 tract cancer. The uniqueness of the epitope may be determined by (i) its
 immunological reactivity and specificity with antibodies directed against
 proteins and polypeptides encoded by SEQUENCE ID NOS 1 through 13, and
 (ii) its nonreactivity with other antibodies directed against other tissue
 markers. Methods for determining immunological reactivity are well-known
 and include, but are not limited to, for example, radioimmunoassay (RIA),
 enzyme-linked immunoabsorbent assay (ELISA), hemagglutination (HA),
 fluorescence polarization immunoassay (FPIA), chemiluminescent immunoassay
 (CLIA) and others. Several examples of suitable methods are described
 herein.
 Unless otherwise stated, the following terms shall have the following
 meanings:
 A polynucleotide "derived from" or "specific for" a designated sequence
 refers to a polynucleotide sequence which comprises a contiguous sequence
 of approximately at least about 6 nucleotides, preferably at least about 8
 nucleotides, more preferably at least about 10-12 nucleotides, and even
 more preferably at least about 15-20 nucleotides corresponding, i.e.,
 identical or complementary to, a region of the designated nucleotide
 sequence. The sequence may be complementary or identical to a sequence
 which is unique to a particular polynucleotide sequence as determined by
 techniques known in the art. Comparisons to sequences in databanks, for
 example, can be used as a method to determine the uniqueness of a
 designated sequence. Regions from which sequences may be derived, include
 but are not limited to, regions encoding specific epitopes, as well as
 non-translated and/or non-transcribed regions.
 The derived polynucleotide will not necessarily be derived physically from
 the nucleotide sequence of interest under study, but may be generated in
 any manner, including, but not limited to, chemical synthesis,
 replication, reverse transcription or transcription, which is based on the
 information provided by the sequence of bases in the region(s) from which
 the polynucleotide is derived. As such, it may represent either a sense or
 an antisense orientation of the original polynucleotide. In addition,
 combinations of regions corresponding to that of the designated sequence
 may be modified in ways known in the art to be consistent with the
 intended use.
 A "fragment" of a specified polynucleotide refers to a polynucleotide
 sequence which comprises a contiguous sequence of approximately at least
 about 6 -nucleotides, preferably at least about 8 nucleotides, more
 preferably at least about 10-12 nucleotides, and even more preferably at
 least about 15-20 nucleotides corresponding, i.e., identical or
 complementary to, a region of the specified nucleotide sequence.
 The term "primer" denotes a specific oligonucleotide sequence which is
 complementary to a target nucleotide sequence and used to hybridize to the
 target nucleotide sequence. A primer serves as an initiation point for
 nucleotide polymerization catalyzed by either DNA polymerase, RNA
 polymerase or reverse transcriptase.
 The term "probe" denotes a defined nucleic acid segment (or nucleotide
 analog segment, e.g., PNA as defined hereinbelow) which can be used to
 identify a specific polynucleotide present in samples bearing the
 complementary sequence. "Encoded by" refers to a nucleic acid sequence
 which codes for a polypeptide sequence, wherein the polypeptide sequence
 or a portion thereof contains an amino acid sequence of at least 3 to 5
 amino acids, more preferably at least 8 to 10 amino acids, and even more
 preferably at least 15 to 20 amino acids from a polypeptide encoded by the
 nucleic acid sequence. Also encompassed are polypeptide sequences which
 are immunologically identifiable with a polypeptide encoded by the
 sequence. Thus, a "polypeptide," "protein," or "amino acid" sequence has
 at least about 50% identity, preferably about 60% identity, more
 preferably about 75-85% identity, and most preferably about 90-95% or more
 identity with an amino acid sequence. Further, the "polypeptide,"
 "protein," or "amino acid" sequence may have at least about 60%
 similarity, preferably at least about 75% similarity, more preferably
 about 85% similarity, and most preferably about 95% or more similarity to
 a polypeptide or amino acid sequence. This amino acid sequence can be
 selected from the group consisting of polypeptides encoded by SEQUENCE ID
 NOS 1 through 13, and fragments thereof.
 A "recombinant polypeptide," "recombinant protein," or "a polypeptide
 produced by recombinant techniques," which terms may be used
 interchangeably herein, describes a polypeptide which by virtue of its
 origin or manipulation is not associated with all or a portion of the
 polypeptide with which it is associated in nature and/or is linked to a
 polypeptide other than that to which it is linked in nature. A recombinant
 or encoded polypeptide or protein is not necessarily translated from a
 designated nucleic acid sequence. It also may be generated in any manner,
 including chemical synthesis or expression of a recombinant expression
 system.
 The term "synthetic peptide" as used herein means a polymeric form of amino
 acids of any length, which may be chemically synthesized by methods
 well-known to the routineer. These synthetic peptides are useful in
 various applications.
 The term "polynucleotide" as used herein means a polymeric form of
 nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
 This term refers only to the primary structure of the molecule. Thus, the
 term includes double- and single-stranded DNA, as well as double- and
 single-stranded RNA. It also includes modifications, such as methylation
 or capping and unmodified forms of the polynucleotide. The terms
 "polynucleotide," "oligomer," "oligonucleotide," and "oligo" are used
 interchangeably herein.
 "A sequence corresponding to a cDNA" means that the sequence contains a
 polynucleotide sequence that is identical or complementary to a sequence
 in the designated DNA. The degree (or "percent") of identity or
 complementarity to the cDNA will be approximately 50% or greater,
 preferably at least about 70% or greater, and more preferably at least
 about 90% or greater. The sequence that corresponds to the identified cDNA
 will be at least about 50 nucleotides in length, preferably at least about
 60 nucleotides in length, and more preferably at least about 70
 nucleotides in length. The correspondence between the gene or gene
 fragment of interest and the cDNA can be determined by methods known in
 the art and include, for example, a direct comparison of the sequenced
 material with the cDNAs described, or hybridization and digestion with
 single strand nucleases, followed by size determination of the digested
 fragments.
 "Purified polynucleotide" refers to a polynucleotide of interest or
 fragment thereof which is essentially free, e.g., contains less than about
 50%, preferably less than about 70%, and more preferably less than about
 90%, of the protein with which the polynucleotide is naturally associated.
 Techniques for purifying polynucleotides of interest are well-known in the
 art and include, for example, disruption of the cell containing the
 polynucleotide with a chaotropic agent and separation of the
 polynucleotide(s) and proteins by ion-exchange chromatography, affinity
 chromatography and sedimentation according to density.
 "Purified polypeptide" or "purified protein" means a polypeptide of
 interest or fragment thereof which is essentially free of, e.g., contains
 less than about 50%, preferably less than about 70%, and more preferably
 less than about 90%, cellular components with which the polypeptide of
 interest is naturally associated. Methods for purifying polypeptides of
 interest are known in the art.
 The term "isolated" means that the material is removed from its original
 environment (e.g., the natural environment if it is naturally occurring).
 For example, a naturally-occurring polynucleotide or polypeptide present
 in a living animal is not isolated, but the same polynucleotide or DNA or
 polypeptide, which is separated from some or all of the coexisting
 materials in the natural system, is isolated. Such polynucleotide could be
 part of a vector and/or such polynucleotide or polypeptide could be part
 of a composition, and still be isolated in that the vector or composition
 is not part of its natural environment.
 "Polypeptide" and "protein" are used interchangeably herein and indicate at
 least one molecular chain of amino acids linked through covalent and/or
 non-covalent bonds. The terms do not refer to a specific length of the
 product. Thus peptides, oligopeptides and proteins are included within the
 definition of polypeptide. The terms include post-translational
 modifications of the polypeptide, for example, glycosylations,
 acetylations, phosphorylations and the like. In addition, protein
 fragments, analogs, mutated or variant proteins, fusion proteins and the
 like are included within the meaning of polypeptide.
 A "fragment" of a specified polypeptide refers to an amino acid sequence
 which comprises at least about 3-5 amino acids, more preferably at least
 about 8-10 amino acids, and even more preferably at least about 15-20
 amino acids derived from the specified polypeptide.
 "Recombinant host cells," "host cells," "cells," "cell lines," "cell
 cultures," and other such terms denoting microorganisms or higher
 eukaryotic cell lines cultured as unicellular entities refer to cells
 which can be, or have been, used as recipients for recombinant vector or
 other transferred DNA, and include the original progeny of the original
 cell which has been transfected.
 As used herein replicon" means any genetic element, such as a plasmid, a
 chromosome or a virus, that behaves as an autonomous unit of
 polynucleotide replication within a cell.
 A "vector" is a replicon in which another polynucleotide segment is
 attached, such as to bring about the replication and/or expression of the
 attached segment.
 The term "control sequence" refers to a polynucleotide sequence which is
 necessary to effect the expression of a coding sequence to which it is
 ligated. The nature of such control sequences differs depending upon the
 host organism. In prokaryotes, such control sequences generally include a
 promoter, a ribosomal binding site and terminators; in eukaryotes, such
 control sequences generally include promoters, terminators and, in some
 instances, enhancers. The term "control sequence" thus is intended to
 include at a minimum all components whose presence is necessary for
 expression, and also may include additional components whose presence is
 advantageous, for example, leader sequences.
 "Operably linked" refers to a situation wherein the components described
 are in a relationship permitting them to function in their intended
 manner. Thus, for example, a control sequence "operably linked" to a
 coding sequence is ligated in such a manner that expression of the coding
 sequence is achieved under conditions compatible with the control
 sequence.
 The term "open reading frame" or "ORF" refers to a region of a
 polynucleotide sequence which encodes a polypeptide. This region may
 represent a portion of a coding sequence or a total coding sequence.
 A "coding sequence" is a polynucleotide sequence which is transcribed into
 mRNA and translated into a polypeptide when placed under the control of
 appropriate regulatory sequences. The boundaries of the coding sequence
 are determined by a translation start codon at the 5'-terminus and a
 translation stop codon at the 3'-terminus. A coding sequence can include,
 but is not limited to, mRNA, cDNA and recombinant polynucleotide
 sequences.
 The term "immunologically identifiable with/as" refers to the presence of
 epitope(s) and polypeptide(s) which also are present in and are unique to
 the designated polypeptide(s). Immunological identity may be determined by
 antibody binding and/or competition in binding. These techniques are known
 to the routineer and also are described herein. The uniqueness of an
 epitope also can be determined by computer searches of known data banks,
 such as GenBank, for the polynucleotide sequence which encodes the epitope
 and by amino acid sequence comparisons with other known proteins.
 As used herein, "epitope" means an antigenic determinant of a polypeptide
 or protein. Conceivably, an epitope can comprise three amino acids in a
 spatial conformation which is unique to the epitope. Generally, an epitope
 consists of at least five such amino acids, and more usually, it consists
 of at least eight to ten amino acids. An epitope can be derived from a
 polypeptide encoded by a nucleic acid molecule described herein. Methods
 of examining spatial conformation are known in the art and include, for
 example, x-ray crystallography and two-dimensional nuclear magnetic
 resonance.
 A "conformational epitope" is an epitope that is comprised of a specific
 juxtaposition of amino acids in an immunologically recognizable structure,
 such amino acids being present on the same polypeptide in a contiguous or
 non-contiguous order or present on different polypeptides.
 A polypeptide is "immunologically reactive" with an antibody when it binds
 to an antibody due to antibody recognition of a specific epitope contained
 within the polypeptide. Immunological reactivity may be determined by
 antibody binding, more particularly, by the kinetics of antibody binding,
 and/or by competition in binding using as competitor(s) a known
 polypeptide(s) containing an epitope against which the antibody is
 directed. The methods for determining whether a polypeptide is
 immunologically reactive with an antibody are known in the art.
 As used herein, the term "immunogenic polypeptide containing an epitope of
 interest" means naturally occurring polypeptides of interest or fragments
 thereof, as well as polypeptides prepared by other means, for example, by
 chemical synthesis or the expression of the polypeptide in a recombinant
 organism.
 The term "transfection" refers to the introduction of an exogenous
 polynucleotide into a prokaryotic or eukaryotic host cell, irrespective of
 the method used for the introduction. The term "transfection" refers to
 both stable and transient introduction of the polynucleotide, and
 encompasses direct uptake of polynucleotides, transformation,
 transduction, and f-mating. Once introduced into the host cell, the
 exogenous polynucleotide may be maintained as a non-integrated replicon,
 for example, a plasmid, or alternatively, may be integrated into the host
 genome.
 "Treatment" refers to prophylaxis and/or therapy.
 The term "individual" as used herein refers to vertebrates, particularly
 members of the mammalian species and includes, but is not limited to,
 domestic animals, sports animals, primates and humans; more particularly,
 the term refers to humans.
 The term "sense strand" or "plus strand" (or "+") as used herein denotes a
 nucleic acid that contains the sequence that encodes the polypeptide. The
 term "antisense strand" or "minus strand" (or "-") denotes a nucleic acid
 that contains a sequence that is complementary to that of the "plus"
 strand.
 The term "test sample" refers to a component of an individual's body which
 is the source of the analyte (such as antibodies of interest or antigens
 of interest). These components are well known in the art. A test sample is
 typically anything suspected of containing a target sequence. Test samples
 can be prepared using methodologies well known in the art such as by
 obtaining a specimen from an individual and, if necessary, disrupting any
 cells contained thereby to release target nucleic acids. These test
 samples include biological samples which can be tested by the methods of
 the present invention described herein and include human and animal body
 fluids such as whole blood, serum, plasma, cerebrospinal fluid, sputum,
 bronchial washing, bronchial aspirates, urine, lymph fluids, and various
 external secretions of the respiratory, intestinal and genitourinary
 tracts, tears, saliva, milk, white blood cells, myelomas and the like;
 biological fluids such as cell culture supernatants; tissue specimens
 which may be fixed; and cell specimens which may be fixed.
 "Purified product" refers to a preparation of the product which has been
 isolated from the cellular constituents with which the product is normally
 associated and from other types of cells which may be present in the
 sample of interest.
 "PNA" denotes a "peptide nucleic acid analog" which may be utilized in a
 procedure such as an assay described herein to determine the presence of a
 target. "MA" denotes a "morpholino analog" which may be utilized in a
 procedure such as an assay described herein to determine the presence of a
 target. See, for example, U.S. Pat. No. 5,378,841, which is incorporated
 herein by reference. PNAs are neutrally charged moieties which can be
 directed against RNA targets or DNA. PNA probes used in assays in place
 of, for example, the DNA probes of the present invention, offer advantages
 not achievable when DNA probes are used. These advantages include
 manufacturability, large scale labeling, reproducibility, stability,
 insensitivity to changes in ionic strength and resistance to enzymatic
 degradation which is present in methods utilizing DNA or RNA. These PNAs
 can be labeled with ("attached to") such signal generating compounds as
 fluorescein, radionucleotides, chemiluminescent compounds and the like.
 PNAs or other nucleic acid analogs such as MAs thus can be used in assay
 methods in place of DNA or RNA. Although assays are described herein
 utilizing DNA probes, it is within the scope of the routineer that PNAs or
 MAs can be substituted for RNA or DNA with appropriate changes if and as
 needed in assay reagents.
 "Analyte," as used herein, is the substance to be detected which may be
 present in the test sample. The analyte can be any substance for which
 there exists a naturally occurring specific binding member (such as an
 antibody), or for which a specific binding member can be prepared. Thus,
 an analyte is a substance that can bind to one or more specific binding
 members in an assay. "Analyte" also includes any antigenic substances,
 haptens, antibodies and combinations thereof. As a member of a specific
 binding pair, the analyte can be detected by means of naturally occurring
 specific binding partners (pairs) such as the use of intrinsic factor
 protein as a member of a specific binding pair for the determination of
 Vitamin B12, the use of folate-binding protein to determine folic acid, or
 the use of a lectin as a member of a specific binding pair for the
 determination of a carbohydrate. The analyte can include a protein, a
 polypeptide, an amino acid, a nucleotide target and the like. The analyte
 can be soluble in a body fluid such as blood, blood plasma or serum, urine
 or the like. The analyte can be in a tissue, either on a cell surface or
 within a cell. The analyte can be on or in a cell dispersed in a body
 fluid such as blood, urine, breast aspirate, or obtained as a biopsy
 sample.
 "Urinary tract cancer," as used herein, refers to any malignant disease of
 the urinary tract including but not limited to, adenocarcinoma,
 transitional cell carcinoma, squamous cell carcinoma, carcinoma in situ,
 clear carcinoma, granular cell carcinoma and sarcomatoid carcinoma.
 An "Expressed Sequence Tag" or "EST" refers to the partial sequence of a
 cDNA insert which has been made by reverse transcription of mRNA extracted
 from a tissue followed by insertion into a vector.
 A "transcript image" refers to a table or list giving the quantitative
 distribution of ESTs in a library and represents the genes active in the
 tissue from which the library was made.
 The present invention provides assays which utilize specific binding
 members. A "specific binding member," as used herein, is a member of a
 specific binding pair. That is, two different molecules where one of the
 molecules, through chemical or physical means, specifically binds to the
 second molecule. Therefore, in addition to antigen and antibody specific
 binding pairs of common immunoassays, other specific binding pairs can
 include biotin and avidin, carbohydrates and lectins, complementary
 nucleotide sequences, effector and receptor molecules, cofactors and
 enzymes, enzyme inhibitors, and enzymes and the like. Furthermore,
 specific binding pairs can include members that are analogs of the
 original specific binding members, for example, an analyte-analog.
 Immunoreactive specific binding members include antigens, antigen
 fragments, antibodies and antibody fragments, both monoclonal and
 polyclonal and complexes thereof, including those formed by recombinant
 DNA molecules.
 Specific binding members include "specific binding molecules." A "specific
 binding molecule" intends any specific binding member, particularly an
 immunoreactive specific binding member. As such, the term "specific
 binding molecule" encompasses antibody molecules (obtained from both
 polyclonal and monoclonal preparations), as well as, the following: hybrid
 (chimeric) antibody molecules (see, for example, Winter, et al., Nature
 349:293-299 (1991), and U.S. Pat. No. 4,816,567); F(ab').sub.2 and F(ab)
 fragments; Fv molecules (non-covalent heterodimers, see, for example,
 Inbar, et al., Proc. Natl. Acad. Sci. USA 69:2659-2662 (1972), and
 Ehrlich, et al., Biochem. 19:4091-4096 (1980)); single chain Fv molecules
 (sFv) (see, for example, Huston, et al., Proc. Natl. Acad. Sci. USA
 85:5879-5883 (1988)); humanized antibody molecules (see, for example,
 Riechmann, et al., Nature 332:323-327 (1988), Verhoeyan, et al., Science
 239:1534-1536 (1988), and UK Patent Publication No. GB 2,276,169,
 published Sep. 21, 1994); and, any functional fragments obtained from such
 molecules, wherein such fragments retain immunological binding properties
 of the parent antibody molecule.
 The term "hapten," as used herein, refers to a partial antigen or
 non-protein binding member which is capable of binding to an antibody, but
 which is not capable of eliciting antibody formation unless coupled to a
 carrier protein.
 A "capture reagent," as used herein, refers to an unlabeled specific
 binding member which is specific either for the analyte as in a sandwich
 assay, for the indicator reagent or analyte as in a competitive assay, or
 for an ancillary specific binding member, which itself is specific for the
 analyte, as in an indirect assay. The capture reagent can be directly or
 indirectly bound to a solid phase material before the performance of the
 assay or during the performance of the assay, thereby enabling the
 separation of immobilized complexes from the test sample.
 The "indicator reagent" comprises a "signal-generating compound" ("label")
 which is capable of generating and generates a measurable signal
 detectable by external means, conjugated ("attached") to a specific
 binding member. In addition to being an antibody member of a specific
 binding pair, the indicator reagent also can be a member of any specific
 binding pair, including either hapten-anti-hapten systems such as biotin
 or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a
 complementary nucleotide sequence, an effector or a receptor molecule, an
 enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme and the
 like. An immunoreactive specific binding member can be an antibody, an
 antigen, or an antibody/antigen complex that is capable of binding either
 to the polypeptide of interest as in a sandwich assay, to the capture
 reagent as in a competitive assay, or to the ancillary specific binding
 member as in an indirect assay. When describing probes and probe assays,
 the term "reporter molecule" may be used. A reporter molecule comprises a
 signal generating compound as described hereinabove conjugated to a
 specific binding member of a specific binding pair, such as carbazole or
 adamantane.
 The various "signal-generating compounds" (labels) contemplated include
 chromagens, catalysts such as enzymes, luminescent compounds such as
 fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes,
 acridiniums, phenanthridiniums and luminol, radioactive elements and
 direct visual labels. Examples of enzymes include alkaline phosphatase,
 horseradish peroxidase, beta-galactosidase and the like. The selection of
 a particular label is not critical, but it must be capable of producing a
 signal either by itself or in conjunction with one or more additional
 substances.
 "Solid phases" ("solid supports") are known to those in the art and include
 the walls of wells of a reaction tray, test tubes, polystyrene beads,
 magnetic or non-magnetic beads, nitrocellulose strips, membranes,
 microparticles such as latex particles, sheep (or other animal) red blood
 cells and Duracytes.RTM. (red blood cells "fixed" by pyruvic aldehyde and
 formaldehyde, available from Abbott Laboratories, Abbott Park, Ill.) and
 others. The "solid phase" is not critical and can be selected by one
 skilled in the art. Thus, latex particles, microparticles, magnetic or
 non-magnetic beads, membranes, plastic tubes, walls of microtiter wells,
 glass or silicon chips, sheep (or other suitable animal's) red blood cells
 and Duracytes.RTM. are all suitable examples. Suitable methods for
 immobilizing peptides on solid phases include ionic, hydrophobic, covalent
 interactions and the like. A "solid phase," as used herein, refers to any
 material which is insoluble, or can be made insoluble by a subsequent
 reaction. The solid phase can be chosen for its intrinsic ability to
 attract and immobilize the capture reagent. Alternatively, the solid phase
 can retain an additional receptor which has the ability to attract and
 immobilize the capture reagent. The additional receptor can include a
 charged substance that is oppositely charged with respect to the capture
 reagent itself or to a charged substance conjugated to the capture
 reagent. As yet another alternative, the receptor molecule can be any
 specific binding member which is immobilized upon (attached to) the solid
 phase and which has the ability to immobilize the capture reagent through
 a specific binding reaction. The receptor molecule enables the indirect
 binding of the capture reagent to a solid phase material before the
 performance of the assay or during the performance of the assay. The solid
 phase thus can be a plastic, derivatized plastic, magnetic or non-magnetic
 metal, glass or silicon surface of a test tube, microtiter well, sheet,
 bead, microparticle, chip, sheep (or other suitable animal's) red blood
 cells, Duracytese and other configurations known to those of ordinary
 skill in the art.
 It is contemplated and within the scope of the present invention that the
 solid phase also can comprise any suitable porous material with sufficient
 porosity to allow access by detection antibodies and a suitable surface
 affinity to bind antigens. Microporous structures generally are preferred,
 but materials with a gel structure in the hydrated state may be used as
 well. Such useful solid supports include, but are not limited to,
 nitrocellulose and nylon. It is contemplated that such porous solid
 supports described herein preferably are in the form of sheets of
 thickness from about 0.01 to 0.5 mm, preferably about 0.1 mm. The pore
 size may vary within wide limits and preferably is from about 0.025 to 15
 microns, especially from about 0.15 to 15 microns. The surface of such
 supports may be activated by chemical processes which cause covalent
 linkage of the antigen or antibody to the support. The irreversible
 binding of the antigen or antibody is obtained, however, in general, by
 adsorption on the porous material by poorly understood hydrophobic forces.
 Other suitable solid supports are known in the art.
 Reagents.
 The present invention provides reagents such as polynucleotide sequences
 represented by SEQUENCE ID NOS 1-13, polypeptides encoded thereby, and
 antibodies specific for these polypeptides. The present invention also
 provides reagents such as oligonucleotide fragments derived from the
 disclosed polynucleotides and nucleic acid sequences complementary to
 these polynucleotides. The polynucleotides, polypeptides, or antibodies of
 the present invention may be used to provide information leading to the
 detecting, diagnosing, staging, monitoring, prognosticating, in vivo
 imaging, preventing or treating of, or determining the predisposition to,
 diseases and conditions of the urinary tract, such as urinary tract
 cancer. The sequences disclosed herein represent unique polynucleotides
 which can be used in assays or for producing a specific profile of gene
 transcription activity. Such assays are disclosed in European Patent
 Number 0373203B1 and International Publication No. WO 95/11995, which are
 hereby incorporated by reference.
 Selected polynucleotides represented by SEQUENCE ID NOS 1-13 can be used in
 the methods described herein for the detection of normal or altered gene
 expression. Such methods may employ polynucleotides or oligonucleotides
 represented by SEQUENCE ID NOS 1-13, fragments or derivatives thereof, or
 nucleic acid sequences complementary thereto.
 The polynucleotides disclosed herein, their complementary sequences, or
 fragments of either, can be used in assays to detect, amplify or quantify
 genes, nucleic acids, cDNAs or mRNAs relating to urinary tract disease and
 conditions associated therewith. They also can be used to identify an
 entire or partial coding region of a polypeptide encoded by SEQUENCE ID
 NOS 1-13. They further can be provided in individual containers in the
 form of a kit for assays, or provided as individual compositions. If
 provided in a kit for assays, other suitable reagents such as buffers,
 conjugates and the like may be included.
 The polynucleotide may be in the form of RNA or DNA. Polynucleotides in the
 form of DNA, cDNA, genomic DNA, nucleic acid analogs and synthetic DNA are
 within the scope of the present invention. The DNA may be double-stranded
 or single-stranded, and if single stranded, may be the coding (sense)
 strand or non-coding (anti-sense) strand. The coding sequence which
 encodes the polypeptide may be identical to the coding sequence provided
 herein or may be a different coding sequence which coding sequence, as a
 result of the redundancy or degeneracy of the genetic code, encodes the
 same polypeptide as the DNA provided herein.
 This polynucleotide may include only the coding sequence for the
 polypeptide, or the coding sequence for the polypeptide and an additional
 coding sequence such as a leader or secretory sequence or a proprotein
 sequence, or the coding sequence for the polypeptide (and optionally an
 additional coding sequence) and non-coding sequence, such as a non-coding
 sequence 5' and/or 3' of the coding sequence for the polypeptide.
 In addition, the invention includes variant polynucleotides containing
 modifications such as polynucleotide deletions, substitutions or
 additions; and any polypeptide modification resulting from the variant
 polynucleotide sequence. A polynucleotide of the present invention also
 may have a coding sequence which is a naturally occurring allelic variant
 of the coding sequence provided herein.
 In addition, the coding sequence for the polypeptide may be fused in the
 same reading frame to a polynucleotide sequence which aids in expression
 and secretion of a polypeptide from a host cell, for example, a leader
 sequence which functions as a secretory sequence for controlling transport
 of a polypeptide from the cell. The polypeptide having a leader sequence
 is a preprotein and may have the leader sequence cleaved by the host cell
 to form the polypeptide. The polynucleotides may also encode for a
 proprotein which is the protein plus additional 5' amino acid residues. A
 protein having a prosequence is a proprotein and may, in some cases, be an
 inactive form of the protein. Once the prosequence is cleaved, an active
 protein remains. Thus, the polynucleotide of the present invention may
 encode for a protein, or for a protein having a prosequence, or for a
 protein having both a presequence (leader sequence) and a prosequence.
 The polynucleotides of the present invention may also have the coding
 sequence fused in frame to a marker sequence which allows for purification
 of the polypeptide of the present invention. The marker sequence may be a
 hexa-histidine tag supplied by a pQE-9 vector to provide for purification
 of the polypeptide fused to the marker in the case of a bacterial host,
 or, for example, the marker sequence may be a hemagglutinin (HA) tag when
 a mammalian host, e.g. a COS-7 cell line, is used. The HA tag corresponds
 to an epitope derived from the influenza hemagglutinin protein. See, for
 example, I. Wilson et al., Cell 37:767 (1984).
 It is contemplated that polynucleotides will be considered to hybridize to
 the sequences provided herein if there is at least 50%, preferably at
 least 70%, and more preferably at least 90% identity between the
 polynucleotide and the sequence.
 The degree of sequence identity between two nucleic acid molecules greatly
 affects the efficiency and strength of hybridization events between such
 molecules. A partially identical nucleic acid sequence is one that will at
 least partially inhibit a completely identical sequence from hybridizing
 to a target molecule. Inhibition of hybridization of the completely
 identical sequence can be assessed using hybridization assays that are
 well known in the art (e.g., Southern blot, Northern blot, solution
 hybridization, in situ hybridization, or the like, see Sambrook, et al.,
 Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring
 Harbor, N.Y.). Such assays can be conducted using varying degrees of
 selectivity, for example, using conditions varying from low to high
 stringency. If conditions of low stringency are employed, the absence of
 non-specific binding can be assessed using a secondary probe that lacks
 even a partial degree of sequence identity (for example, a probe having
 less than about 30% sequence identity with the target molecule), such
 that, in the absence of non-specific binding events, the secondary probe
 will not hybridize to the target.
 When utilizing a hybridization-based detection system, a nucleic acid probe
 is chosen that is complementary to a target nucleic acid sequence, and
 then by selection of appropriate conditions the probe and the target
 sequence "selectively hybridize," or bind, to each other to form a hybrid
 molecule. In one embodiment of the present invention, a nucleic acid
 molecule is capable of hybridizing selectively to a target sequence under
 moderately stringent hybridization conditions. In the context of the
 present invention, moderately stringent hybridization conditions allow
 detection of a target nucleic acid sequence of at least 14 nucleotides in
 length having at least approximately 70% sequence identity with the
 sequence of the selected nucleic acid probe. In another embodiment, such
 selective hybridization is performed under stringent hybridization
 conditions. Stringent hybridization conditions allow detection of target
 nucleic acid sequences of at least 14 nucleotides in length having a
 sequence identity of greater than 90% with the sequence of the selected
 nucleic acid probe. Hybridization conditions useful for probe/target
 hybridization where the probe and target have a specific degree of
 sequence identity, can be determined as is known in the art (see, for
 example, Nucleic Acid Hybridization: A Practical Approach, editors B. D.
 Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).
 Hybrid molecules can be formed, for example, on a solid support, in
 solution, and in tissue sections. The formation of hybrids can be
 monitored by inclusion of a reporter molecule, typically, in the probe.
 Such reporter molecules, or detectable elements include, but are not
 limited to, radioactive elements, fluorescent markers, and molecules to
 which an enzyme-conjugated ligand can bind.
 With respect to stringency conditions for hybridization, it is well known
 in the art that numerous equivalent conditions can be employed to
 establish a particular stringency by varying, for example, the following
 factors: the length and nature of probe and target sequences, base
 composition of the various sequences, concentrations of salts and other
 hybridization solution components, the presence or absence of blocking
 agents in the hybridization solutions (e.g., formamide, dextran sulfate,
 and polyethylene glycol), hybridization reaction temperature and time
 parameters, as well as, varying wash conditions. The selection of a
 particular set of hybridization conditions is well within the skill of the
 routineer in the art (see, for example, Sambrook, et al., Molecular
 Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor,
 N.Y.).
 The present invention also provides an antibody produced by using a
 purified polypeptide encoded by a marker represented by SEQUENCE ID NOS
 1-13 of which at least a portion of the polypeptide is encoded by a
 polynucleotide represented by SEQUENCE ID NOS 1-13. These antibodies may
 be used in the methods provided herein for the detection of antigen
 encoded by markers represented by SEQUENCE ID NOS 1-13 in test samples.
 The presence of antigen encoded by markers represented by SEQUENCE ID NOS
 1-13 in the test samples is indicative of the presence of a urinary tract
 disease or condition, including urinary tract cancer. The antibody also
 may be used for therapeutic purposes, for example, in neutralizing the
 activity of polypeptide encoded by markers represented by SEQUENCE ID NOS
 1-13 in conditions associated with altered or abnormal expression.
 The present invention further relates to a polypeptide encoded by markers
 represented by SEQUENCE ID NOS 1-13 which has the deduced amino acid
 sequence as provided herein, as well as fragments, analogs and derivatives
 of such polypeptide. The polypeptide of the present invention may be a
 recombinant polypeptide, a natural purified polypeptide or a synthetic
 polypeptide. The fragment, derivative or analog of the polypeptide encoded
 by markers represented by SEQUENCE ID NOS 1-13 may be one in which one or
 more of the amino acid residues is substituted with a conserved or
 non-conserved amino acid residue (preferably a conserved amino acid
 residue) and such substituted amino acid residue may or may not be one
 encoded by the genetic code; or it may be one in which one or more of the
 amino acid residues includes a substituent group; or it may be one in
 which the polypeptide is fused with another compound, such as a compound
 to increase the half-life of the polypeptide (for example, polyethylene
 glycol); or it may be one in which the additional amino acids are fused to
 the polypeptide, such as a leader or secretory sequence or a sequence
 which is employed for purification of the polypeptide or a proprotein
 sequence. Such fragments, derivatives and analogs are within the scope of
 the present invention. The polypeptides and polynucleotides of the present
 invention are provided preferably in an isolated form and preferably
 purified.
 Thus, a polypeptide of the present invention may have an amino acid
 sequence that is identical to that of the naturally occurring polypeptide
 or that is different by minor variations due to one or more amino acid
 substitutions. The variation may be a "conservative change" typically in
 the range of about 1 to 5 amino acids, wherein the substituted amino acid
 has similar structural or chemical properties, e.g., replacement of
 leucine with isoleucine or threonine with serine. In contrast, variations
 may include nonconservative changes, e.g., replacement of a glycine with a
 tryptophan. Similar minor variations may also include amino acid deletions
 or insertions, or both. Guidance in determining which and how many amino
 acid residues may be substituted, inserted or deleted without changing
 biological or immunological activity may be found using computer programs
 well known in the art, for example, DNASTAR software (DNASTAR Inc.,
 Madison Wis.).
 Probes constructed according to the polynucleotide sequences of the present
 invention can be used in various assay methods to provide various types of
 analysis. For example, such probes can be used in fluorescent in situ
 hybridization (FISH) technology to perform chromosomal analysis, and used
 to identify cancer-specific structural alterations in the chromosomes,
 such as deletions or translocations that are visible from chromosome
 spreads or detectable using PCR-generated and/or allele specific
 oligonucleotides probes, allele specific amplification or by direct
 sequencing. Probes also can be labeled with radioisotopes, directly- or
 indirectly-detectable haptens, or fluorescent molecules, and utilized for
 in situ hybridization studies to evaluate the mRNA expression of the gene
 comprising the polynucleotide in tissue specimens or cells.
 This invention also provides teachings as to the production of the
 polynucleotides and polypeptides provided herein.
 Probe Assavs.
 The sequences provided herein may be used to produce probes which can be
 used in assays for the detection of nucleic acids in test samples. The
 probes may be designed from conserved nucleotide regions of the
 polynucleotides of interest or from non-conserved nucleotide regions of
 the polynucleotide of interest. The design of such probes for optimization
 in assays is within the skill of the routineer. Generally, nucleic acid
 probes are developed from non-conserved or unique regions when maximum
 specificity is desired, and nucleic acid probes are developed from
 conserved regions when assaying for nucleotide regions that are closely
 related to, for example, different members of a multi-gene family or in
 related species like mouse and man.
 The polymerase chain reaction (PCR) is a technique for amplifying a desired
 nucleic acid sequence (target) contained in a nucleic acid or mixture
 thereof. In PCR, a pair of primers are employed in excess to hybridize to
 the complementary strands of the target nucleic acid. The primers are each
 extended by a polymerase using the target nucleic acid as a template. The
 extension products become target sequences themselves, following
 dissociation from the original target strand. New primers then are
 hybridized and extended by a polymerase, and the cycle is repeated to
 geometrically increase the number of target sequence molecules. PCR is
 disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202, which are
 incorporated herein by reference.
 The Ligase Chain Reaction (LCR) is an alternate method for nucleic acid
 amplification. In LCR, probe pairs are used which include two primary
 (first and second) and two secondary (third and fourth) probes, all of
 which are employed in molar excess to target. The first probe hybridizes
 to a first segment of the target strand, and the second probe hybridizes
 to a second segment of the target strand, the first and second segments
 being contiguous so that the primary probes abut one another in 5'
 phosphate-3' hydroxyl relationship, and so that a ligase can covalently
 fuse or ligate the two probes into a fused product. In addition, a third
 (secondary) probe can hybridize to a portion of the first probe and a
 fourth (secondary) probe can hybridize to a portion of the second probe in
 a similar abutting fashion. Of course, if the target is initially double
 stranded, the secondary probes also will hybridize to the target
 complement in the first instance. Once the ligated strand of primary
 probes is separated from the target strand, it will hybridize with the
 third and fourth probes which can be ligated to form a complementary,
 secondary ligated product. It is important to realize that the ligated
 products are functionally equivalent to either the target or its
 complement. By repeated cycles of hybridization and ligation,
 amplification of the target sequence is achieved. This technique is
 described more completely in EP-A-320 308 to K. Backman published Jun. 16,
 1989 and EP-A-439 182 to K. Backman et al., published Jul. 31, 1991, both
 of which are incorporated herein by reference.
 For amplification of mRNAs, it is within the scope of the present invention
 to reverse transcribe mRNA into cDNA followed by polymerase chain reaction
 (RT-PCR); or, to use a single enzyme for both steps as described in U.S.
 Pat. No. 5,322,770, which is incorporated herein by reference; or reverse
 transcribe mRNA into cDNA followed by asymmetric gap ligase chain reaction
 (RT-AGLCR) as described by R. L. Marshall et al., PCR Methods and
 Applications 4:80-84 (1994), which also is incorporated herein by
 reference.
 Other known amplification methods which can be utilized herein include but
 are not limited to the so-called "NASBA" or "3SR" technique described by
 J. C. Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878 (1990) and
 also described by J. Compton, Nature 350 (No. 6313):91-92 (1991); Q-beta
 amplification as described in published European Patent Application (EPA)
 No. 4544610; strand displacement amplification (as described in G. T.
 Walker et al., Clin. Chem. 42:9-13 [1996]) and European Patent Application
 No. 684315; and target mediated amplification, as described in
 International Publication No. WO 93/22461.
 Detection of markers represented by SEQUENCE ID NOS 1-13 may be
 accomplished using any suitable detection method, including those
 detection methods which are currently well known in the art, as well as
 detection strategies which may evolve later. Examples of the foregoing
 presently known detection methods are hereby incorporated herein by
 reference. See, for example, Caskey et al., U.S. Pat. No. 5,582,989,
 Gelfand et al., U.S. Pat. No. 5,210,015. Examples of such detection
 methods include target amplification methods as well as signal
 amplification technologies. An example of presently known detection
 methods would include the nucleic acid amplification technologies referred
 to as PCR, LCR, NASBA, SDA, RCR and TMA. See, for example, Caskey et al.,
 U.S. Pat. No. 5,582,989, Gelfand et al., U.S. Pat. No. 5,210,015. All of
 the foregoing are hereby incorporated by reference. Detection may also be
 accomplished using signal amplification such as that disclosed in Snitman
 et al., U.S. Pat. No. 5,273,882. While the amplification of target or
 signal is preferred at present, it is contemplated and within the scope of
 the present invention that ultrasensitive detection methods which do not
 require amplification can be utilized herein.
 Detection, both amplified and non-amplified, may be performed using a
 variety of heterogeneous and homogeneous detection formats. Examples of
 heterogeneous detection formats are disclosed in Snitman et al., U.S. Pat.
 No. 5,273,882, Albarella et al in EP-84114441.9, Urdea et al., U.S. Pat.
 No. 5,124,246, Ullman et al. U.S. Pat. No. 5,185,243 and Kourilsky et al.,
 U.S. Pat. No. 4,581,333. All of the foregoing are hereby incorporated by
 reference. Examples of homogeneous detection formats are disclosed in,
 Caskey et al., U.S. Pat. No. 5,582,989, Gelfand et al., U.S. Pat. No.
 5,210,015, which are incorporated herein by reference. Also contemplated
 and within the scope of the present invention is the use of multiple
 probes in the hybridization assay, which use improves sensitivity and
 amplification of the signal. See, for example, Caskey et al., U.S. Pat.
 No. 5,582,989, Gelfand et al., U.S. Pat. No. 5,210,015, which are
 incorporated herein by reference.
 In one embodiment, the present invention generally comprises the steps of
 contacting a test sample suspected of containing a target polynucleotide
 sequence with amplification reaction reagents comprising an amplification
 primer, and a detection probe that can hybridize with an internal region
 of the amplicon sequences. Probes and primers employed according to the
 method provided herein are labeled with capture and detection labels,
 wherein probes are labeled with one type of label and primers are labeled
 with another type of label. Additionally, the primers and probes are
 selected such that the probe sequence has a lower melt temperature than
 the primer sequences. The amplification reagents, detection reagents and
 test sample are placed under amplification conditions whereby, in the
 presence of target sequence, copies of the target sequence (an amplicon)
 are produced. In the usual case, the amplicon is double stranded because
 primers are provided to amplify a target sequence and its complementary
 strand. The double stranded amplicon then is thermally denatured to
 produce single stranded amplicon members. Upon formation of the single
 stranded amplicon members, the mixture is cooled to allow the formation of
 complexes between the probes and single stranded amplicon members.
 As the single stranded amplicon sequences and probe sequences are cooled,
 the probe sequences preferentially bind the single stranded amplicon
 members. This finding is counterintuitive given that the probe sequences
 generally are selected to be shorter than the primer sequences and
 therefore have a lower melt temperature than the primers. Accordingly, the
 melt temperature of the amplicon produced by the primers should also have
 a higher melt temperature than the probes. Thus, as the mixture cools, the
 re-formation of the double stranded amplicon would be expected. As
 previously stated, however, this is not the case. The probes are found to
 preferentially bind the single stranded amplicon members. Moreover, this
 preference of probe/single stranded amplicon binding exists even when the
 primer sequences are added in excess of the probes.
 After the probe/single stranded amplicon member hybrids are formed, they
 are detected. Standard heterogeneous assay formats are suitable for
 detecting the hybrids using the detection labels and capture labels
 present on the primers and probes. The hybrids can be bound to a solid
 phase reagent by virtue of the capture label and detected by virtue of the
 detection label. In cases where the detection label is directly
 detectable, the presence of the hybrids on the solid phase can be detected
 by causing the label to produce a detectable signal, if necessary, and
 detecting the signal. In cases where the label is not directly detectable,
 the captured hybrids can be contacted with a conjugate, which generally
 comprises a binding member attached to a directly detectable label. The
 conjugate becomes bound to the complexes and the conjugate's presence on
 the complexes can be detected with the directly detectable label. Thus,
 the presence of the hybrids on the solid phase reagent can be determined.
 Those skilled in the art will recognize that wash steps may be employed to
 wash away unhybridized amplicon or probe as well as unbound conjugate.
 In one embodiment, the heterogeneous assays can be conveniently performed
 using a solid phase support that carries an array of nucleic acid
 molecules. Such arrays are useful for high-throughput and/or multiplexed
 assay formats. Various methods for forming such arrays from pre-formed
 nucleic acid molecules, or methods for generating the array using in situ
 synthesis techniques, are generally known in the art. (See, for example,
 Dattagupta, et al., EP Publication No. 0 234, 726A3; Southern, U.S. Pat.
 No. 5,700,637; Pirrung, et al., U.S. Pat. No. 5,143,854; PCT International
 Publication No. WO 92/10092; and, Fodor, et al., Science 251:767-777
 (1991)).
 Although the target sequence is described as single stranded, it also is
 contemplated to include the case where the target sequence is actually
 double stranded but is merely separated from its complement prior to
 hybridization with the amplification primer sequences. In the case where
 PCR is employed in this method, the ends of the target sequences are
 usually known. In cases where LCR or a modification thereof is employed in
 the preferred method, the entire target sequence is usually known.
 Typically, the target sequence is a nucleic acid sequence such as, for
 example, RNA or DNA.
 The method provided herein can be used in well-known amplification
 reactions that include thermal cycle reaction mixtures, particularly in
 PCR and gap LCR (GLCR). Amplification reactions typically employ primers
 to repeatedly generate copies of a target nucleic acid sequence, which
 target sequence is usually a small region of a much larger nucleic acid
 sequence. Primers are themselves nucleic acid sequences that are
 complementary to regions of a target sequence. Under amplification
 conditions, these primers hybridize or bind to the complementary regions
 of the target sequence. Copies of the target sequence typically are
 generated by the process of primer extension and/or ligation which
 utilizes enzymes with polymerase or ligase activity, separately or in
 combination, to add nucleotides to the hybridized primers and/or ligate
 adjacent probe pairs. The nucleotides that are added to the primers or
 probes, as monomers or preformed oligomers, are also complementary to the
 target sequence. Once the primers or probes have been sufficiently
 extended and/or ligated, they are separated from the target sequence, for
 example, by heating the reaction mixture to a "melt temperature" which is
 one in which complementary nucleic acid strands dissociate. Thus, a
 sequence complementary to the target sequence is formed.
 A new amplification cycle then can take place to further amplify the number
 of target sequences by separating any double stranded sequences, allowing
 primers or probes to hybridize to their respective targets, extending
 and/or ligating the hybridized primers or probes and re-separating. The
 complementary sequences that are generated by amplification cycles can
 serve as templates for primer extension or filling the gap of two probes
 to further amplify the number of target sequences. Typically, a reaction
 mixture is cycled between 20 and 100 times, more typically, a reaction
 mixture is cycled between 25 and 50 times. The numbers of cycles can be
 determined by the routineer. In this manner, multiple copies of the target
 sequence and its complementary sequence are produced. Thus, primers
 initiate amplification of the target sequence when it is present under
 amplification conditions.
 Generally, two primers which are complementary to a portion of a target
 strand and its complement are employed in PCR. For LCR, four probes, two
 of which are complementary to a target sequence and two of which are
 similarly complementary to the target's complement, are generally
 employed. In addition to the primer sets and enzymes previously mentioned,
 a nucleic acid amplification reaction mixture may also comprise other
 reagents which are well known and include but are not limited to: enzyme
 cofactors such as manganese; magnesium; salts; nicotinamide adenine
 dinucleotide (NAD); and deoxynucleotide triphosphates (dNTPs) such as, for
 example, deoxyadenine triphosphate, deoxyguanine triphosphate,
 deoxycytosine triphosphate and deoxythymine triphosphate.
 While the amplification primers initiate amplification of the target
 sequence, the detection (or hybridization) probe is not involved in
 amplification. Detection probes are generally nucleic acid sequences or
 uncharged nucleic acid analogs such as, for example, peptide nucleic acids
 which are disclosed in International Publication No. WO 92/20702;
 morpholino analogs which are described in U.S. Pat. Nos. 5,185,444,
 5,034,506 and 5,142,047; and the like. Depending upon the type of label
 carried by the probe, the probe is employed to capture or detect the
 amplicon generated by the amplification reaction. The probe is not
 involved in amplification of the target sequence and therefore may have to
 be rendered "non-extendible" in that additional dNTPs cannot be added to
 the probe. In and of themselves, analogs usually are non-extendible and
 nucleic acid probes can be rendered non-extendible by modifying the 3' end
 of the probe such that the hydroxyl group is no longer capable of
 participating in elongation. For example, the 3' end of the probe can be
 functionalized with the capture or detection label to thereby consume or
 otherwise block the hydroxyl group. Alternatively, the 3' hydroxyl group
 simply can be cleaved, replaced or modified. U.S. patent application Ser.
 No. 07/049,061 filed Apr. 19, 1993 and incorporated herein by reference
 describes modifications which can be used to render a probe
 non-extendible.
 The ratio of primers to probes is not important. Thus, either the probes or
 primers can be added to the reaction mixture in excess whereby the
 concentration of one would be greater than the concentration of the other.
 Alternatively, primers and probes can be employed in equivalent
 concentrations. Preferably, however, the primers are added to the reaction
 mixture in excess of the probes. Thus, primer to probe ratios of, for
 example, 5:1 and 20: 1, are preferred.
 While the length of the primers and probes can vary, the probe sequences
 are selected such that they have a lower melt temperature than the primer
 sequences. Hence, the primer sequences are generally longer than the probe
 sequences. Typically, the primer sequences are in the range of between 20
 and 50 nucleotides long, more typically in the range of between 20 and 30
 nucleotides long. The typical probe is in the range of between 10 and 25
 nucleotides long.
 Various methods for synthesizing primers and probes are well known in the
 art. Similarly, methods for attaching labels to primers or probes are also
 well known in the art. For example, it is a matter of routine to
 synthesize desired nucleic acid primers or probes using conventional
 nucleotide phosphoramidite chemistry and instruments available from
 Applied Biosystems, Inc., (Foster City, Calif.), DuPont (Wilmington,
 Del.), or Milligen (Bedford Mass.). Many methods have been described for
 labeling oligonucleotides such as the primers or probes of the present
 invention. Enzo Biochemical (New York, N.Y.) and Clontech (Palo Alto,
 Calif.) both have described and commercialized probe labeling techniques.
 For example, a primary amine can be attached to a 3' oligo terminus using
 3'-Amine-ON CPGTM (Clontech, Palo Alto, Calif.). Similarly, a primary
 amine can be attached to a 5' oligo terminus using Aminomodifier IIO
 (Clontech). The amines can be reacted to various haptens using
 conventional activation and linking chemistries. In addition, copending
 applications U.S. Ser. No. 625,566, filed Dec. 11, 1990 and Ser. No.
 630,908, filed Dec. 20, 1990, which are each incorporated herein by
 reference, teach methods for labeling probes at their 5' and 3' termini,
 respectively. International Publication Nos WO 92/10505, published Jun.
 25, 1992, and WO 92/11388, published Jul. 9, 1992, teach methods for
 labeling probes at their 5' and 3' ends, respectively. According to one
 known method for labeling an oligonucleotide, a label-phosphoramidite
 reagent is prepared and used to add the label to the oligonucleotide
 during its synthesis. See, for example, N. T. Thuong et al., Tet. Letters
 29(46):5905-5908 (1988); or J. S. Cohen et al., published U.S. patent
 application Ser. No. 07/246,688 (NTIS ORDER No. PAT-APPL-7-246,688)
 (1989). Preferably, probes are labeled at their 3' and 5' ends.
 A capture label is attached to the primers or probes and can be a specific
 binding member which forms a binding pair with the solid phase reagent's
 specific binding member. It will be understood that the primer or probe
 itself may serve as the capture label. For example, in the case where a
 solid phase reagent's binding member is a nucleic acid sequence, it may be
 selected such that it binds a complementary portion of the primer or probe
 to thereby immobilize the primer or probe to the solid phase. In cases
 where the probe itself serves as the binding member, those skilled in the
 art will recognize that the probe will contain a sequence or "tail" that
 is not complementary to the single stranded amplicon members. In the case
 where the primer itself serves as the capture label, at least a portion of
 the primer will be free to hybridize with a nucleic acid on a solid phase
 because the probe is selected such that it is not fully complementary to
 the primer sequence.
 Generally, probe/single stranded amplicon member complexes can be detected
 using techniques commonly employed to perform heterogeneous immunoassays.
 Preferably, in this embodiment, detection is performed according to the
 protocols used by the commercially available Abbott LCx.RTM.
 instrumentation (Abbott Laboratories, Abbott Park, Ill.).
 The primers and probes disclosed herein are useful in typical PCR assays,
 wherein the test sample is contacted with a pair of primers, amplification
 is performed, the hybridization probe is added, and detection is
 performed.
 Another method provided by the present invention comprises contacting a
 test sample with a plurality of polynucleotides, wherein at least one
 polynucleotide is a molecule represented by SEQUENCE ID NOS 1-13 as
 described herein, hybridizing the test sample with the plurality of
 polynucleotides and detecting hybridization complexes. Hybridization
 complexes are identified and quantitated to compile a profile which is
 indicative of urinary tract disease, such as urinary tract cancer.
 Expressed RNA sequences may further be detected by reverse transcription
 and amplification of the DNA product by procedures well-known in the art,
 including polymerase chain reaction (PCR).
 Drug Screening and Gene Therapy.
 The present invention also encompasses the use of gene therapy methods for
 the introduction of anti-sense molecules represented by SEQUENCE ID NOS
 1-13 derived molecules, such as polynucleotides or oligonucleotides of the
 present invention, into patients with conditions associated with abnormal
 expression of polynucleotides related to a urinary tract disease or
 condition especially urinary tract cancer. These molecules, including
 antisense RNA and DNA fragments and ribozymes, are designed to inhibit the
 translation of mRNA represented by SEQUENCE ID NOS 1-13, and may be used
 therapeutically in the treatment of conditions associated with altered or
 abnormal expression of polynucleotide represented by SEQUENCE ID NOS 1-13.
 Alternatively, the oligonucleotides described above can be delivered to
 cells by procedures known in the art such that the anti-sense RNA or DNA
 may be expressed in vivo to inhibit production of a polypeptide encoded by
 markers represented by SEQUENCE ID NOS 1-13 in the manner described above.
 Antisense constructs to a polynucleotide represented by SEQUENCE ID NOS
 1-13, therefore, reverse the action of transcripts represented by SEQUENCE
 ID NOS 1-13 and may be used for treating urinary tract disease conditions,
 such as urinary tract cancer. These antisense constructs may also be used
 to treat tumor metastases.
 The present invention also provides a method of screening a plurality of
 compounds for specific binding to polypeptide(s) encoded by markers
 represented by SEQUENCE ID NOS 1-13, or any fragment thereof, to identify
 at least one compound which specifically binds the polypeptide encoded by
 markers represented by SEQUENCE ID NOS 1-13. Such a method comprises the
 steps of providing at least one compound; combining the polypeptide
 encoded by markers represented by SEQUENCE ID NOS 1-13 with each compound
 under suitable conditions for a time sufficient to allow binding; and
 detecting the polypeptide encoded by markers represented by SEQUENCE ID
 NOS 1-13 binding to each compound.
 The polypeptide or peptide fragment employed in such a test may either be
 free in solution, affixed to a solid support, borne on a cell surface or
 located intracellularly. One method of screening utilizes eukaryotic or
 prokaryotic host cells which are stably transfected with recombinant
 nucleic acids which can express the polypeptide or peptide fragment. A
 drug, compound, or any other agent may be screened against such
 transfected cells in competitive binding assays. For example, the
 formation of complexes between a polypeptide and the agent being tested
 can be measured in either viable or fixed cells.
 The present invention thus provides methods of screening for drugs,
 compounds, or any other agent which can be used to treat diseases
 associated with markers represented by SEQUENCE ID NOS 1-13. These methods
 comprise contacting the agent with a polypeptide or fragment thereof and
 assaying for either the presence of a complex between the agent and the
 polypeptide, or for the presence of a complex between the polypeptide and
 the cell. In competitive binding assays, the polypeptide typically is
 labeled. After suitable incubation, free (or uncomplexed) polypeptide or
 fragment thereof is separated from that present in bound form, and the
 amount of free or uncomplexed label is used as a measure of the ability of
 the particular agent to bind to the polypeptide or to interfere with the
 polypeptide/cell complex.
 The present invention also encompasses the use of competitive screening
 assays in which neutralizing antibodies capable of binding polypeptide
 specifically compete with a test agent for binding to the polypeptide or
 fragment thereof. In this manner, the antibodies can be used to detect the
 presence of any polypeptide in the test sample which shares one or more
 antigenic determinants with a polypeptide encoded by markers represented
 by SEQUENCE ID NOS 1-13 as provided herein.
 Another technique for screening provides high throughput screening for
 compounds having suitable binding affinity to at least one polypeptide
 encoded by markers represented by SEQUENCE ID NOS 1-13 disclosed herein.
 Briefly, large numbers of different small peptide test compounds are
 synthesized on a solid phase, such as plastic pins or some other surface.
 The peptide test compounds are reacted with polypeptide and washed.
 Polypeptide thus bound to the solid phase is detected by methods
 well-known in the art. Purified polypeptide can also be coated directly
 onto plates for use in the screening techniques described herein. In
 addition, non-neutralizing antibodies can be used to capture the
 polypeptide and immobilize it on the solid support. See, for example, EP
 84/03564, published on September 13, 1984, which is incorporated herein by
 reference.
 The goal of rational drug design is to produce structural analogs of
 biologically active polypeptides of interest or of the small molecules
 including agonists, antagonists, or inhibitors with which they interact.
 Such structural analogs can be used to design drugs which are more active
 or stable forms of the polypeptide or which enhance or interfere with the
 function of a polypeptide in vivo. J. Hodgson, Bio/Technology 9:19-21
 (1991), incorporated herein by reference.
 For example, in one approach, the three-dimensional structure of a
 polypeptide, or of a polypeptide-inhibitor complex, is determined by x-ray
 crystallography, by computer modeling or, most typically, by a combination
 of the two approaches. Both the shape and charges of the polypeptide must
 be ascertained to elucidate the structure and to determine active site(s)
 of the molecule. Less often, useful information regarding the structure of
 a polypeptide may be gained by modeling based on the structure of
 homologous proteins. In both cases, relevant structural information is
 used to design analogous polypeptide-like molecules or to identify
 efficient inhibitors
 Useful examples of rational drug design may include molecules which have
 improved activity or stability as shown by S. Braxton et al., Biochemistry
 31:7796-7801 (1992), or which act as inhibitors, agonists, or antagonists
 of native peptides as shown by S. B. P. Athauda et al., J Biochem. (Tokyo)
 113 (6):742-746 (1993), incorporated herein by reference.
 It also is possible to isolate a target-specific antibody selected by an
 assay as described hereinabove, and then to determine its crystal
 structure. In principle this approach yields a pharmacophore upon which
 subsequent drug design can be based. It further is possible to bypass
 protein crystallography altogether by generating anti-idiotypic antibodies
 ("anti-ids") to a functional, pharmacologically active antibody. As a
 mirror image of a mirror image, the binding site of the anti-id is an
 analog of the original receptor. The anti-id then can be used to identify
 and isolate peptides from banks of chemically or biologically produced
 peptides. The isolated peptides then can act as the pharmacophore (that
 is, a prototype pharmaceutical drug).
 A sufficient amount of a recombinant polypeptide of the present invention
 may be made available to perform analytical studies such as X-ray
 crystallography. In addition, knowledge of the polypeptide amino acid
 sequence which is derivable from the nucleic acid sequence provided herein
 will provide guidance to those employing computer modeling techniques in
 place of, or in addition to, x-ray crystallography.
 Antibodies specific to a polypeptide encoded by markers represented by
 SEQUENCE ID NOS 1-13 (e.g., anti-marker antibodies) further may be used to
 inhibit the biological action of the polypeptide by binding to the
 polypeptide. In this manner, the antibodies may be used in therapy, for
 example, to treat urinary tract diseases including urinary tract cancer
 and its metastases.
 Further, such antibodies can detect the presence or absence of a
 polypeptide encoded by markers represented by SEQUENCE ID NOS 1-13 in a
 test sample and, therefore, are useful as diagnostic markers for the
 diagnosis of a urinary tract disease or condition especially urinary tract
 cancer. Such antibodies may also function as a diagnostic marker for
 urinary tract disease conditions, such as urinary tract cancer.
 The present invention also is directed to antagonists and inhibitors of the
 polypeptides of the present invention. The antagonists and inhibitors are
 those which inhibit or eliminate the function of the polypeptide. Thus,
 for example, an antagonist may bind to a polypeptide of the present
 invention and inhibit or eliminate its function. The antagonist, for
 example, could be an antibody against the polypeptide which eliminates the
 activity of a polypeptide encoded by markers represented by SEQUENCE ID
 NOS 1-13 by binding a polypeptide encoded by markers represented by
 SEQUENCE ID NOS 1-13, or in some cases the antagonist may be an
 oligonucleotide. Examples of small molecule inhibitors include, but are
 not limited to, small peptides or peptide-like molecules.
 The antagonists and inhibitors may be employed as a composition with a
 pharmaceutically acceptable carrier including, but not limited to, saline,
 buffered saline, dextrose, water, glycerol, ethanol and combinations
 thereof. Administration of polypeptide inhibitors is preferably systemic.
 The present invention also provides an antibody which inhibits the action
 of such a polypeptide.
 Antisense technology can be used to reduce gene expression through
 triple-helix formation or antisense DNA or RNA, both of which methods are
 based on binding of a polynucleotide to DNA or RNA. For example, the 5'
 coding portion of the polynucleotide sequence, which encodes for the
 polypeptide of the present invention, is used to design an antisense RNA
 oligonucleotide of from 10 to 40 base pairs in length. A DNA
 oligonucleotide is designed to be complementary to a region of the gene
 involved in transcription, thereby preventing transcription and the
 production of the polypeptide encoded by markers represented by SEQUENCE
 ID NOS 1-13. For triple helix, see, for example, Lee et al., Nuc. Acids
 Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et
 al., Science 251:1360 (1991) The antisense RNA oligonucleotide hybridizes
 to the mRNA in vivo and blocks translation of a mRNA molecule into the
 polypeptide encoded by markers represented by SEQUENCE ID NOS 1-13. For
 antisense, see, for example, Okano, J. Neurochem. 56:560 (1991); and
 Oligodeoxvnucleotides as Antisense Inhibitors of Gene Expression, CRC
 Press, Boca Raton, Fla. (1988). Antisense oligonucleotides act with
 greater efficacy when modified to contain artificial intemucleotide
 linkages which render the molecule resistant to nucleolytic cleavage. Such
 artificial internucleotide linkages include, but are not limited to,
 methylphosphonate, phosphorothiolate and phosphoroamydate internucleotide
 linkages.
 Recombinant Technology.
 The present invention provides host cells and expression vectors comprising
 polynucleotides represented by SEQUENCE ID NOS 1-13 of the present
 invention and methods for the production of the polypeptide(s) they
 encode. Such methods comprise culturing the host cells under conditions
 suitable for the expression of the polynucleotide represented by SEQUENCE
 ID NOS 1-13 and recovering the encoded polypeptide from the cell culture.
 The present invention also provides vectors which include polynucleotides
 represented by SEQUENCE ID NOS 1-13 of the present invention, host cells
 which are genetically engineered with vectors of the present invention and
 the production of polypeptides of the present invention by recombinant
 techniques.
 Host cells are genetically engineered (transfected, transduced or
 transformed) with the vectors of this invention which may be cloning
 vectors or expression vectors. The vector may be in the form of a plasmid,
 a viral particle, a phage, etc. The engineered host cells can be cultured
 in conventional nutrient media modified as appropriate for activating
 promoters, selecting transfected cells, or amplifying gene(s) represented
 by SEQUENCE ID NOS 1-13. The culture conditions, such as temperature, pH
 and the like, are those previously used with the host cell selected for
 expression, and will be apparent to the ordinarily skilled artisan.
 The polynucleotides of the present invention may be employed for producing
 a polypeptide by recombinant techniques. Thus, the polynucleotide sequence
 may be included in any one of a variety of expression vehicles, in
 particular, vectors or plasmids for expressing a polypeptide. Such vectors
 include chromosomal, nonchromosomal and synthetic DNA sequences, e.g.,
 derivatives of SV40; bacterial plasmids; phage DNA; yeast plasmids;
 vectors derived from combinations of plasmids and phage DNA, viral DNA
 such as vaccinia, adenovirus, fowl pox virus and pseudorabies. However,
 any other plasmid or vector may be used so long as it is replicable and
 viable in the host.
 The appropriate DNA sequence may be inserted into the vector by a variety
 of procedures. In general, the DNA sequence is inserted into appropriate
 restriction endonuclease sites by procedures known in the art. Such
 procedures and others are deemed to be within the scope of those skilled
 in the art. The DNA sequence in the expression vector is operatively
 linked to an appropriate expression control sequence(s) (promoter) to
 direct mRNA synthesis. Representative examples of such promoters include,
 but are not limited to, the LTR or the SV40 promoter, the E. coli lac or
 trp, the phage lambda P sub L promoter and other promoters known to
 control expression of genes in prokaryotic or eukaryotic cells or their
 viruses. The expression vector also contains a ribosome binding site for
 translation initiation and a transcription terminator. The vector may also
 include appropriate sequences for amplifying expression. In addition, the
 expression vectors preferably contain a gene to provide a phenotypic trait
 for selection of transfected host cells such as dihydrofolate reductase or
 neomycin resistance for eukaryotic cell culture, or such as tetracycline
 or ampicillin resistance in E. coli.
 The vector containing the appropriate DNA sequence as hereinabove
 described, as well as an appropriate promoter or control sequence, may be
 employed to transfect an appropriate host to permit the host to express
 the protein. As representative examples of appropriate hosts, there may be
 mentioned: bacterial cells, such as E. coli, Salmonella typhimurium;
 Streptomyces sp; fungal cells, such as yeast; insect cells, such as
 Drosophila and Sf9; animal cells, such as CHO, COS or Bowes melanoma;
 plant cells, etc. The selection of an appropriate host is deemed to be
 within the scope of those skilled in the art from the teachings provided
 herein.
 More particularly, the present invention also includes recombinant
 constructs comprising one or more of the sequences as broadly described
 above. The constructs comprise a vector, such as a plasmid or viral
 vector, into which a sequence of the invention has been inserted, in a
 forward or reverse orientation. In a preferred aspect of this embodiment,
 the construct further comprises regulatory sequences including, for
 example, a promoter, operably linked to the sequence. Large numbers of
 suitable vectors and promoters are known to those of skill in the art and
 are commercially available. The following vectors are provided by way of
 example. Bacterial: pINCY (Incyte Pharmaceuticals Inc., Palo Alto,
 Calif.), pSPORT1 (Life Technologies, Gaithersburg, Md.), pQE70, pQE60,
 pQE-9 (Qiagen) pBs, phagescript, psiXI74, pBluescript SK, pBsKS, pNH8a,
 pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540,
 pRIT5 (Pharmacia); Eukaryotic: pWLneo, pSV2cat, pOG44, pXTI, pSG
 (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other
 plasmid or vector may be used as long as it is replicable and viable in
 the host.
 Plasmid pINCY is generally identical to the plasmid pSPORT I (available
 from Life Technologies, Gaithersburg, Md.) with the exception that it has
 two modifications in the polylinker (multiple cloning site). These
 modifications are (1) it lacks a HindIII restriction site and (2) its
 EcoRI restriction site lies at a different location. pINCY is created from
 pSPORT1 by cleaving pSPORT1 with both HindIfI and EcoRI and replacing the
 excised fragment of the polylinker with synthetic DNA fragments. This
 replacement may be made in any manner known to those of ordinary skill in
 the art. For example, the two nucleotide sequences, may be generated
 synthetically with 5' terminal phosphates, mixed together, and then
 ligated under standard conditions for performing staggered end ligations
 into the pSPORT1 plasmid cut with HindIII and EcoRI. Suitable host cells
 (such as E. coli DH5.mu. cells) then are transfected with the ligated DNA
 and recombinant clones are selected for ampicillin resistance. Plasmid DNA
 then is prepared from individual clones and subjected to restriction
 enzyme analysis or DNA sequencing in order to confirm the presence of
 insert sequences in the proper orientation. Other cloning strategies known
 to the ordinary artisan also may be employed.
 Promoter regions can be selected from any desired gene using CAT
 (chloramphenicol transferase) vectors or other vectors with selectable
 markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named
 bacterial promoters include lacd, lacZ, T3, SP6, T7, gpt, lambda P sub R,
 P sub L and trp. Eukaryotic promoters include cytomegalovirus (CMV)
 immediate early, herpes simplex virus (HSV) thymidine kinase, early and
 late SV40, LTRs from retroviruses and mouse metallothionein-I. Selection
 of the appropriate vector and promoter is well within the level of
 ordinary skill in the art.
 In a further embodiment, the present invention provides host cells
 containing the above-described construct. The host cell can be a higher
 eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,
 such as a yeast cell, or the host cell can be a prokaryotic cell, such as
 a bacterial cell. Introduction of the construct into the host cell can be
 effected by calcium phosphate transfection, DEAE-Dextran mediated
 transfection, or electroporation [L. Davis et al., Basic Methods in
 Molecular Biology, 2nd edition, Appleton and Lang, Paramount Publishing,
 East Norwalk, Conn. (1994)].
 The constructs in host cells can be used in a conventional manner to
 produce the gene product encoded by the recombinant sequence.
 Alternatively, the polypeptides of the invention can be synthetically
 produced by conventional peptide synthesizers.
 Recombinant proteins can be expressed in mammalian cells, yeast, bacteria,
 or other cells, under the control of appropriate promoters. Cell-free
 translation systems can also be employed to produce such proteins using
 RNAs derived from the DNA constructs of the present invention. Appropriate
 cloning and expression vectors for use with prokaryotic and eukaryotic
 hosts are described by Sambrook et al., Molecular Cloning: A Laboratory
 Manual, Second Edition, (Cold Spring Harbor, N.Y., 1989), which is hereby
 incorporated by reference.
 Transcription of a DNA encoding the polypeptide(s) of the present invention
 by higher eukaryotes is increased by inserting an enhancer sequence into
 the vector. Enhancers are cis-acting elements of DNA, usually about from
 10 to 300 bp, that act on a promoter to increase its transcription.
 Examples include the SV40 enhancer on the late side of the replication
 origin (bp 100 to 270), a cytomegalovirus early promoter enhancer, a
 polyoma enhancer on the late side of the replication origin and adenovirus
 enhancers.
 Generally, recombinant expression vectors will include origins of
 replication and selectable markers permitting transfection of the host
 cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae
 TRP 1 gene, and a promoter derived from a highly-expressed gene to direct
 transcription of a downstream structural sequence. Such promoters can be
 derived from operons encoding glycolytic enzymes such as
 3-phosphoglycerate kinase (PGK), alpha factor, acid phosphatase, or heat
 shock proteins, among others. The heterologous structural sequence is
 assembled in appropriate phase with translation initiation and termination
 sequences, and preferably, a leader sequence capable of directing
 secretion of translated protein into the periplasmic space or
 extracellular medium. Optionally, the heterologous sequence can encode a
 fusion protein including an N-terminal identification peptide imparting
 desired characteristics, e.g., stabilization or simplified purification of
 expressed recombinant product.
 Useful expression vectors for bacterial use are constructed by inserting a
 structural DNA sequence encoding a desired protein together with suitable
 translation initiation and termination signals in operable reading phase
 with a functional promoter. The vector will comprise one or more
 phenotypic selectable markers and an origin of replication to ensure
 maintenance of the vector and to, if desirable, provide amplification
 within the host. Suitable prokaryotic hosts for transfection include E.
 coli, Bacillus subtilis, Salmonella typhimurium and various species within
 the genera Pseudomonas, Streptomyces and Staphylococcus, although others
 may also be employed as a routine matter of choice.
 Useful expression vectors for bacterial use comprise a selectable marker
 and bacterial origin of replication derived from plasmids comprising
 genetic elements of the well-known cloning vector pBR322 (ATCC 37017).
 Other vectors include but are not limited to PKK223-3 (Pharmacia Fine
 Chemicals, Uppsala, Sweden) and GEMI (Promega Biotec, Madison, Wis.).
 These pBR322 "backbone" sections are combined with an appropriate promoter
 and the structural sequence to be expressed.
 Following transfection of a suitable host and growth of the host to an
 appropriate cell density, the selected promoter is derepressed by
 appropriate means (e.g., temperature shift or chemical induction), and
 cells are cultured for an additional period. Cells are typically harvested
 by centrifugation, disrupted by physical or chemical means, and the
 resulting crude extract retained for further purification. Microbial cells
 employed in expression of proteins can be disrupted by any convenient
 method including freeze-thaw cycling, sonication, mechanical disruption,
 or use of cell lysing agents. Such methods are well-known to the ordinary
 artisan.
 Various mammalian cell culture systems can also be employed to express
 recombinant protein. Examples of mammalian expression systems include the
 COS-7 lines of monkey kidney fibroblasts described by Gluzman, Cell 23:175
 (1981), and other cell lines capable of expressing a compatible vector,
 such as the C127, HEK-293, 3T3, CHO, HeLa and BHK cell lines. Mammalian
 expression vectors will comprise an origin of replication, a suitable
 promoter and enhancer and also any necessary ribosome binding sites,
 polyadenylation sites, splice donor and acceptor sites, transcriptional
 termination sequences and 5' flanking nontranscribed sequences. DNA
 sequences derived from the SV40 viral genome, for example, SV40 origin,
 early promoter, enhancer, splice, and polyadenylation sites may be used to
 provide the required nontranscribed genetic elements. Representative,
 useful vectors include pRc/CMV and pcDNA3 (available from Invitrogen, San
 Diego, Calif.).
 Polypeptides encoded by markers represented by SEQUENCE ID NOS 1-13 are
 recovered and purified from recombinant cell cultures by known methods
 including affinity chromatography, ammonium sulfate or ethanol
 precipitation, acid extraction, anion or cation exchange chromatography,
 phosphocellulose chromatography, hydrophobic interaction chromatography,
 hydroxyapatite chromatography or lectin chromatography. It is preferred to
 have low concentrations (approximately 0.1-5 mM) of calcium ion present
 during purification [Price, et al., J. Biol. Chem. 244:917 (1969)].
 Protein refolding steps can be used, as necessary, in completing
 configuration of the polypeptide. Finally, high performance liquid
 chromatography (HPLC) can be employed for final purification steps.
 Thus, polypeptides of the present invention may be naturally purified
 products expressed from a high expressing cell line, or a product of
 chemical synthetic procedures, or produced by recombinant techniques from
 a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher
 plant, insect and mammalian cells in culture). Depending upon the host
 employed in a recombinant production procedure, the polypeptides of the
 present invention may be glycosylated with mammalian or other eukaryotic
 carbohydrates or may be non-glycosylated. The polypeptides of the
 invention may also include an initial methionine amino acid residue.
 The starting plasmids can be constructed from available plasmids in accord
 with published, known procedures. In addition, equivalent plasmids to
 those described are known in the art and will be apparent to one of
 ordinary skill in the art.
 Methods for DNA sequencing are well known in the art. Conventional
 enzymatic methods employ DNA polymerase, Klenow fragment, Sequenase (US
 Biochemical Corp, Cleveland, Ohio) or Taq polymerase to extend DNA chains
 from an oligonucleotide primer annealed to the DNA template of interest.
 Methods have been developed for the use of both single-stranded and
 double-stranded templates. The chain termination reaction products may be
 electrophoresed on urea/polyacrylamide gels and detected either by
 autoradiography (for radionucleotide labeled precursors) or by
 fluorescence (for fluorescent-labeled precursors). Recent improvements in
 mechanized reaction preparation, sequencing and analysis using the
 fluorescent detection method have permitted expansion in the number of
 sequences that can be determined per day using machines such as the
 Applied Biosystems 377 DNA Sequencers (Applied Biosystems, Foster City,
 Calif.).
 The reading frame of the nucleotide sequence can be ascertained by several
 types of analyses. First, reading frames contained within the coding
 sequence can be analyzed for the presence of start codon ATG and stop
 codons TGA, TAA or TAG. Typically, one reading frame will continue
 throughout the major portion of a cDNA sequence while other reading frames
 tend to contain numerous stop codons. In such cases, reading frame
 determination is straightforward. In other more difficult cases, further
 analysis is required.
 Algorithms have been created to analyze the occurrence of individual
 nucleotide bases at each putative codon triplet. See, for example J. W.
 Fickett, Nuc. Acids Res. 10:5303 (1982). Coding DNA for particular
 organisms (bacteria, plants and animals) tends to contain certain
 nucleotides within certain triplet periodicities, such as a significant
 preference for pyrimidines in the third codon position. These preferences
 have been incorporated into widely available software which can be used to
 determine coding potential (and frame) of a given stretch of DNA. The
 algorithm-derived information combined with start/stop codon information
 can be used to determine proper frame with a high degree of certainty.
 This, in turn, readily permits cloning of the sequence in the correct
 reading frame into appropriate expression vectors.
 The nucleic acid sequences disclosed herein may be joined to a variety of
 other polynucleotide sequences and vectors of interest by means of
 well-established recombinant DNA techniques. See J. Sambrook et al.,
 supra. Vectors of interest include cloning vectors, such as plasmids,
 cosmids, phage derivatives, phagemids, as well as sequencing, replication
 and expression vectors, and the like. In general, such vectors contain an
 origin of replication functional in at least one organism, convenient
 restriction endonuclease digestion sites and selectable markers
 appropriate for particular host cells. The vectors can be transferred by a
 variety of means known to those of skill in the art into suitable host
 cells which then produce the desired DNA, RNA or polypeptides.
 Occasionally, sequencing or random reverse transcription errors will mask
 the presence of the appropriate open reading frame or regulatory element.
 In such cases, it is possible to determine the correct reading frame by
 attempting to express the polypeptide and determining the amino acid
 sequence by standard peptide mapping and sequencing techniques. See, F. M.
 Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons,
 New York, N.Y. (1989). Additionally, the actual reading frame of a given
 nucleotide sequence may be determined by transfection of host cells with
 vectors containing all three potential reading frames. Only those cells
 with the nucleotide sequence in the correct reading frame will produce a
 peptide of the predicted length.
 The nucleotide sequences provided herein have been prepared by current,
 state-of-the-art, automated methods and, as such, may contain unidentified
 nucleotides. These will not present a problem to those skilled in the art
 who wish to practice the invention. Several methods employing standard
 recombinant techniques, described in J. Sambrook (supra) or periodic
 updates thereof, may be used to complete the missing sequence information.
 The same techniques used for obtaining a full length sequence, as
 described herein, may be used to obtain nucleotide sequences.
 Expression of a particular cDNA may be accomplished by subcloning the cDNA
 into an appropriate expression vector and transfecting this vector into an
 appropriate expression host. The cloning vector used for the generation of
 the urinary tract tissue cDNA library can be used for transcribing mRNA of
 a particular cDNA and contains a promoter for beta-galactosidase, an
 amino-terminal met and the subsequent seven amino acid residues of
 beta-galactosidase. Immediately following these eight residues is an
 engineered bacteriophage promoter useful for artificial priming and
 transcription, as well as a number of unique restriction sites, including
 EcoRi, for cloning. The vector can be transfected into an appropriate host
 strain of E. coli.
 Induction of the isolated bacterial strain with isopropylthiogalactoside
 (IPTG) using standard methods will produce a fusion protein which contains
 the first seven residues of beta-galactosidase, about 15 residues of
 linker and the peptide encoded within the cDNA. Since cDNA clone inserts
 are generated by an essentially random process, there is one chance in
 three that the included cDNA will lie in the correct frame for proper
 translation. If the cDNA is not in the proper reading frame, the correct
 frame can be obtained by deletion or insertion of an appropriate number of
 bases by well known methods including in vitro mutagenesis, digestion with
 exonuclease III or mung bean nuclease, or oligonucleotide linker
 inclusion.
 The cDNA can be shuttled into other vectors known to be useful for
 expression of protein in specific hosts. Oligonucleotide primers
 containing cloning sites and segments of DNA sufficient to hybridize to
 stretches at both ends of the target cDNA can be synthesized chemically by
 standard methods. These primers can then be used to amplify the desired
 gene segments by PCR. The resulting new gene segments can be digested with
 appropriate restriction enzymes under standard conditions and isolated by
 gel electrophoresis. Alternately, similar gene segments can be produced by
 digestion of the cDNA with appropriate restriction enzymes and filling in
 the missing gene segments with chemically synthesized oligonucleotides.
 Segments of the coding sequence from more than one gene can be ligated
 together and cloned in appropriate vectors to optimize expression of
 recombinant sequence.
 Suitable expression hosts for such chimeric molecules include, but are not
 limited to, mammalian cells, such as Chinese Hamster Ovary (CHO) and human
 embryonic kidney (HEK) 293 cells, insect cells, such as Sf9 cells, yeast
 cells, such as Saccharomyces cerevisiae and bacteria, such as E. coli. For
 each of these cell systems, a useful expression vector may also include an
 origin of replication to allow propagation in bacteria and a selectable
 marker such as the beta-lactamase antibiotic resistance gene to allow
 selection in bacteria. In addition, the vectors may include a second
 selectable marker, such as the neomycin phosphotransferase gene, to allow
 selection in transfected eukaryotic host cells. Vectors for use in
 eukaryotic expression hosts may require the addition of 3' poly A tail if
 the sequence of interest lacks poly A.
 Additionally, the vector may contain promoters or enhancers which increase
 gene expression. Such promoters are host specific and include, but are not
 limited to, MMTV, SV40, or metallothionine promoters for CHO cells; trp,
 lac, tac or T7 promoters for bacterial hosts; or alpha factor, alcohol
 oxidase or PGH promoters for yeast. Adenoviral vectors with or without
 transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,
 may be used to drive protein expression in mammalian cell lines. Once
 homogeneous cultures of recombinant cells are obtained, large quantities
 of recombinantly produced protein can be recovered from the conditioned
 medium and analyzed using chromatographic methods well known in the art.
 An alternative method for the production of large amounts of secreted
 protein involves the transfection of mammalian embryos and the recovery of
 the recombinant protein from milk produced by transgenic cows, goats,
 sheep, etc. Polypeptides and closely related molecules may be expressed
 recombinantly in such a way as to facilitate protein purification. One
 approach involves expression of a chimeric protein which includes one or
 more additional polypeptide domains not naturally present on human
 polypeptides. Such purification-facilitating domains include, but are not
 limited to, metal-chelating peptides such as histidine-tryptophan domains
 that allow purification on immobilized metals, protein A domains that
 allow purification on immobilized immunoglobulin and the domain utilized
 in the FLAGS extension/affinity purification system (Immunex Corp,
 Seattle, Wash.). The inclusion of a cleavable linker sequence such as
 Factor XA or enterokinase from Invitrogen (San Diego, Calif.) between the
 polypeptide sequence and the purification domain may be useful for
 recovering the polypeptide.
 Immunoassays.
 Polypeptides encoded by markers represented by SEQUENCE ID NOS 1-13,
 including fragments, derivatives, and analogs thereof, or cells expressing
 such polypeptides, can be utilized in a variety of assays, many of which
 are described herein, for the detection of antibodies to urinary tract
 tissue. They also can be used as immunogens to produce antibodies. These
 antibodies can be, for example, polyclonal or monoclonal antibodies,
 chimeric, single chain and humanized antibodies, as well as Fab fragments,
 or the product of an Fab expression library. Various procedures known in
 the art may be used for the production of such antibodies and fragments.
 For example, antibodies generated against a polypeptide comprising a
 sequence of the present invention can be obtained by direct injection of
 the polypeptide into an animal or by administering the polypeptide to an
 animal such as a mouse, rabbit, goat or human. A mouse, rabbit or goat is
 preferred. The polypeptide is encoded by markers represented by SEQUENCE
 ID NOS 1-13, and fragments thereof. The antibody so obtained then will
 bind the polypeptide itself. In this manner, even a sequence encoding only
 a fragment of the polypeptide can be used to generate antibodies that bind
 the native polypeptide. Such antibodies then can be used to isolate the
 polypeptide from test samples such as tissue suspected of containing that
 polypeptide. For preparation of monoclonal antibodies, any technique which
 provides antibodies produced by continuous cell line cultures can be used.
 Examples include the hybridoma technique as described by Kohler and
 Milstein, Nature 256:495-497 (1975), the trioma technique, the human
 B-cell hybridoma technique as described by Kozbor et al., Immun. Today
 4:72 (1983) and the EBV-hybridoma technique to produce human monoclonal
 antibodies as described by Cole et al., in Monoclonal Antibodies and
 Cancer Therapy, Alan R. Liss, Inc, New York, N.Y., pp. 77-96 (1985).
 Techniques described for the production of single chain antibodies can be
 adapted to produce single chain antibodies to immunogenic polypeptide
 products of this invention. See, for example, U.S. Pat. No. 4,946,778,
 which is incorporated herein by reference.
 Various assay formats may utilize the antibodies of the present invention,
 including "sandwich" immunoassays and probe assays. For example, the
 antibodies of the present invention, or fragments thereof, can be employed
 in various assay systems to determine the presence, if any, of antigen
 encoded by markers represented by SEQUENCE ID NOS 1-13 in a test sample.
 For example, in a first assay format, a polyclonal or monoclonal antibody
 or fragment thereof, or a combination of these antibodies, which has been
 coated on a solid phase, is contacted with a test sample, to form a first
 mixture. This first mixture is incubated for a time and under conditions
 sufficient to form antigen/antibody complexes. Then, an indicator reagent
 comprising a monoclonal or a polyclonal antibody or a fragment thereof, or
 a combination of these antibodies, to which a signal generating compound
 has been attached, is contacted with the antigen/antibody complexes to
 form a second mixture. This second mixture then is incubated for a time
 and under conditions sufficient to form antibody/antigen/antibody
 complexes. The presence of antigen encoded by markers represented by
 SEQUENCE ID NOS 1-13 in the test sample and captured on the solid phase,
 if any, is determined by detecting the measurable signal generated by the
 signal generating compound. The amount of antigen encoded by markers
 represented by SEQUENCE ID NOS 1-13 present in the test sample is
 proportional to the signal generated.
 In an alternative assay format, a mixture is formed by contacting: (1) a
 polyclonal antibody, monoclonal antibody, or fragment thereof, which
 specifically binds to antigen encoded by markers represented by SEQUENCE
 ID NOS 1-13, or a combination of such antibodies bound to a solid support;
 (2) the test sample; and (3) an indicator reagent comprising a monoclonal
 antibody, polyclonal antibody, or fragment thereof, which specifically
 binds to a different antigen encoded by markers represented by SEQUENCE ID
 NOS 1-13 (or a combination of these antibodies) to which a signal
 generating compound is attached. This mixture is incubated for a time and
 under conditions sufficient to form antibody/antigen/antibody complexes.
 The presence, if any, of antigen encoded by markers represented by
 SEQUENCE ID NOS 1-13 present in the test sample and captured on the solid
 phase is determined by detecting the measurable signal generated by the
 signal generating compound. The amount of antigen encoded by markers
 represented by SEQUENCE ID NOS 1-13 present in the test sample is
 proportional to the signal generated.
 In another assay format, one or a combination of at least two monoclonal
 antibodies of the invention can be employed as a competitive probe for the
 detection of antibodies to antigen encoded by markers represented by
 SEQUENCE ID NOS 1-13. For example, polypeptides such as the recombinant
 antigens disclosed herein, either alone or in combination, are coated on a
 solid phase. A test sample suspected of containing antibody to antigen
 encoded by markers represented by SEQUENCE ID NOS 1-13 then is incubated
 with an indicator reagent comprising a signal generating compound and at
 least one monoclonal antibody of the invention for a time and under
 conditions sufficient to form antigen/antibody complexes of either the
 test sample and indicator reagent bound to the solid phase or the
 indicator reagent bound to the solid phase. The reduction in binding of
 the monoclonal antibody to the solid phase can be quantitatively measured.
 In yet another detection method, each of the monoclonal or polyclonal
 antibodies of the present invention can be employed in the detection of
 antigens encoded by markers represented by SEQUENCE ID NOS 1-13 in tissue
 sections, as well as in cells, by immunohistochemical analysis. The tissue
 sections can be cut from either frozen or chemically fixed samples of
 tissue. If the antigens are to be detected in cells, the cells can be
 isolated from blood, urine, breast aspirates, or other bodily fluids. The
 cells may be obtained by biopsy, either surgical or by needle. The cells
 can be isolated by centrifugation or magnetic attraction after labeling
 with magnetic particles or ferrofluids so as to enrich a particular
 fraction of cells for staining with the antibodies of the present
 invention. Cytochemical analysis wherein these antibodies are labeled
 directly (with, for example, fluorescein, colloidal gold, horseradish
 peroxidase, alkaline phosphatase, etc.) or are labeled by using secondary
 labeled anti-species antibodies (with various labels as exemplified
 herein) to track the histopathology of disease also are within the scope
 of the present invention.
 In addition, these monoclonal antibodies can be bound to matrices similar
 to CNBr-activated Sepharose and used for the affinity purification of
 specific polypeptides encoded by markers represented by SEQUENCE ID NOS
 1-13 from cell cultures or biological tissues such as to purify
 recombinant and native proteins encoded by markers represented by SEQUENCE
 ID NOS 1-13.
 The monoclonal antibodies of the invention also can be used for the
 generation of chimeric antibodies for therapeutic use, or other similar
 applications.
 The monoclonal antibodies or fragments thereof can be provided individually
 to detect antigens encoded by markers represented by SEQUENCE ID NOS 1-13.
 Combinations of the monoclonal antibodies (and fragments thereof) provided
 herein also may be used together as components in a mixture or "cocktail"
 of at least one antibody of the invention, along with antibodies which
 specifically bind to other regions, each antibody having different binding
 specificities. Thus, this cocktail can include the monoclonal antibodies
 of the invention which are directed to polypeptides encoded by markers
 represented by SEQUENCE ID NOS 1-13 disclosed herein and other monoclonal
 antibodies specific to other antigenic determinants of antigens encoded by
 markers represented by SEQUENCE ID NOS 1-13 or other related proteins.
 The polyclonal antibody or fragment thereof which can be used in the assay
 formats should specifically bind to a polypeptide encoded by markers
 represented by SEQUENCE ID NOS 1-13 or other polypeptides encoded by
 markers represented by SEQUENCE ID NOS 1-13 additionally used in the
 assay. The polyclonal antibody used preferably is of mammalian origin such
 as, human, goat, rabbit or sheep polyclonal antibody which binds
 polypeptide encoded by markers represented by SEQUENCE ID NOS 1-13. Most
 preferably, the polyclonal antibody is of rabbit origin. The polyclonal
 antibodies used in the assays can be used either alone or as a cocktail of
 polyclonal antibodies. Since the cocktails used in the assay formats are
 comprised of either monoclonal antibodies or polyclonal antibodies having
 different binding specificity to polypeptides encoded by markers
 represented by SEQUENCE ID NOS 1-13, they are useful for the detecting,
 diagnosing, staging, monitoring, prognosticating, in vivo imaging,
 preventing or treating, or determining the predisposition to, diseases and
 conditions of the urinary tract, such as urinary tract cancer.
 It is contemplated and within the scope of the present invention that
 antigen encoded by markers represented by SEQUENCE ID NOS 1-13 may be
 detectable in assays by use of a recombinant antigen as well as by use of
 a synthetic peptide or purified peptide, which peptide comprises an amino
 acid sequence of markers represented by SEQUENCE ID NOS 1-13. The amino
 acid sequence of such a polypeptide is selected from the group consisting
 of those encoded by markers represented by SEQUENCE ID NOS 1-13, and
 fragments thereof. It also is within the scope of the present invention
 that different synthetic, recombinant or purified peptides, identifying
 different epitopes of markers represented by SEQUENCE ID NOS 1-13, can be
 used in combination in an assay for the detecting, diagnosing, staging,
 monitoring, prognosticating, in vivo imaging, preventing or treating, or
 determining the predisposition to diseases and conditions of the urinary
 tract, such as urinary tract cancer. In this case, all of these peptides
 can be coated onto one solid phase; or each separate peptide may be coated
 onto separate solid phases, such as microparticles, and then combined to
 form a mixture of peptides which can be later used in assays. Furthermore,
 it is contemplated that multiple peptides which define epitopes from
 different antigens may be used for the detection, diagnosis, staging,
 monitoring, prognosis, prevention or treatment of, or determining the
 predisposition to, diseases and conditions of the urinary tract, such as
 urinary tract cancer. Peptides coated on solid phases or labeled with
 detectable labels are then allowed to compete with those present in a
 patient sample (if any) for a limited amount of antibody. A reduction in
 binding of the synthetic, recombinant, or purified peptides to the
 antibody (or antibodies) is an indication of the presence of antigen
 encoded by markers represented by SEQUENCE ID NOS 1-13 in the patient
 sample. The presence of antigen encoded by markers represented by SEQUENCE
 ID NOS 1-13 indicates the presence of urinary tract disease, especially
 urinary tract cancer, in the patient. Variations of assay formats are
 known to those of ordinary skill in the art and many are discussed herein
 below.
 In another assay format, the presence of antibodies against markers
 represented by SEQUENCE ID NOS 1-13 and/or antigen encoded by markers
 represented by SEQUENCE ID NOS 1-13 can be detected in a simultaneous
 assay, as follows. A test sample is simultaneously contacted with a
 capture reagent of a first analyte, wherein said capture reagent comprises
 a first binding member specific for a first analyte attached to a solid
 phase and a capture reagent for a second analyte, wherein said capture
 reagent comprises a first binding member for a second analyte attached to
 a second solid phase, to thereby form a mixture. This mixture is incubated
 for a time and under conditions sufficient to form capture reagent/first
 analyte and capture reagent/second analyte complexes. These so-formed
 complexes then are contacted with an indicator reagent comprising a member
 of a binding pair specific for the first analyte labeled with a signal
 generating compound and an indicator reagent comprising a member of a
 binding pair specific for the second analyte labeled with a signal
 generating compound to form a second mixture. This second mixture is
 incubated for a time and under conditions sufficient to form capture
 reagent/first analyte/indicator reagent complexes and capture
 reagent/second analyte/indicator reagent complexes. The presence of one or
 more analytes is determined by detecting a signal generated in connection
 with the complexes formed on either or both solid phases as an indication
 of the presence of one or more analytes in the test sample. In this assay
 format, recombinant antigens derived from the expression systems disclosed
 herein may be utilized, as well as monoclonal antibodies produced from the
 proteins derived from the expression systems as disclosed herein. For
 example, in this assay system, antigen encoded by markers represented by
 SEQUENCE ID NOS 1-13 can be the first analyte. Such assay systems are
 described in greater detail in EP Publication No. 0473065.
 In yet other assay formats, the polypeptides disclosed herein may be
 utilized to detect the presence of antibody against antigen encoded by
 markers represented by SEQUENCE ID NOS 1-13 in test samples. For example,
 a test sample is incubated with a solid phase to which at least one
 polypeptide such as a recombinant protein or synthetic peptide has been
 attached. The polypeptide is selected from the group consisting of those
 encoded by markers represented by SEQUENCE ID NOS 1-13, and fragments
 thereof. These are reacted for a time and under conditions sufficient to
 form antigen/antibody complexes. Following incubation, the
 antigen/antibody complex is detected. Indicator reagents may be used to
 facilitate detection, depending upon the assay system chosen. In another
 assay format, a test sample is contacted with a solid phase to which a
 recombinant protein produced as described herein is attached, and also is
 contacted with a monoclonal or polyclonal antibody specific for the
 protein, which preferably has been labeled with an indicator reagent.
 After incubation for a time and under conditions sufficient for
 antibody/antigen complexes to form, the solid phase is separated from the
 free phase, and the label is detected in either the solid or free phase as
 an indication of the presence of antibody against antigen encoded by
 markers represented by SEQUENCE ID NOS 1-13. Other assay formats utilizing
 the recombinant antigens disclosed herein are contemplated. These include
 contacting a test sample with a solid phase to which at least one antigen
 from a first source has been attached, incubating the solid phase and test
 sample for a time and under conditions sufficient to form antigen/antibody
 complexes, and then contacting the solid phase with a labeled antigen,
 which antigen is derived from a second source different from the first
 source. For example, a recombinant protein derived from a first source
 such as E. coli is used as a capture antigen on a solid phase, a test
 sample is added to the so-prepared solid phase, and following standard
 incubation and washing steps as deemed or required, a recombinant protein
 derived from a different source (i.e., non-E. coli) is utilized as a part
 of an indicator reagent which subsequently is detected. Likewise,
 combinations of a recombinant antigen on a solid phase and synthetic
 peptide in the indicator phase also are possible. Any assay format which
 utilizes an antigen specific for markers represented by SEQUENCE ID NOS
 1-13 produced or derived from a first source as the capture antigen and an
 antigen specific for markers represented by SEQUENCE ID NOS 1-13 from a
 different second source is contemplated. Thus, various combinations of
 recombinant antigens, as well as the use of synthetic peptides, purified
 proteins and the like, are within the scope of this invention. Assays such
 as this and others are described in U.S. Pat. No. 5,254,458, which enjoys
 common ownership and is incorporated herein by reference.
 Other embodiments which utilize various other solid phases also are
 contemplated and are within the scope of this invention. For example, ion
 capture procedures for immobilizing an immobilizable reaction complex with
 a negatively charged polymer (described in EP publication 0326100 and EP
 publication No. 0406473), can be employed according to the present
 invention to effect a fast solution-phase immunochemical reaction. An
 immobilizable immune complex is separated from the rest of the reaction
 mixture by ionic interactions between the negatively charged
 poly-anion/immune complex and the previously treated, positively charged
 porous matrix and detected by using various signal generating systems
 previously described, including those described in chemiluminescent signal
 measurements as described in EPO Publication No. 0 273,115.
 Also, the methods of the present invention can be adapted for use in
 systems which utilize microparticle technology including automated and
 semi-automated systems wherein the solid phase comprises a microparticle
 (magnetic or non-magnetic). Such systems include those described in, for
 example, published EPO applications Nos. EP 0 425 633 and EP 0 424 634,
 respectively.
 The use of scanning probe microscopy (SPM) for immunoassays also is a
 technology to which the monoclonal antibodies of the present invention are
 easily adaptable. In scanning probe microscopy, particularly in atomic
 force microscopy, the capture phase, for example, at least one of the
 monoclonal antibodies of the invention, is adhered to a solid phase and a
 scanning probe microscope is utilized to detect antigen/antibody complexes
 which may be present on the surface of the solid phase. The use of
 scanning tunneling microscopy eliminates the need for labels which
 normally must be utilized in many immunoassay systems to detect
 antigen/antibody complexes. The use of SPM to monitor specific binding
 reactions can occur in many ways. In one embodiment, one member of a
 specific binding partner (analyte specific substance which is the
 monoclonal antibody of the invention) is attached to a surface suitable
 for scanning. The attachment of the analyte specific substance may be by
 adsorption to a test piece which comprises a solid phase of a plastic or
 metal surface, following methods known to those of ordinary skill in the
 art. Or, covalent attachment of a specific binding partner (analyte
 specific substance) to a test piece which test piece comprises a solid
 phase of derivatized plastic, metal, silicon, or glass may be utilized.
 Covalent attachment methods are known to those skilled in the art and
 include a variety of means to irreversibly link specific binding partners
 to the test piece. If the test piece is silicon or glass, the surface must
 be activated prior to attaching the specific binding partner. Also,
 polyelectrolyte interactions may be used to immobilize a specific binding
 partner on a surface of a test piece by using techniques and chemistries.
 The preferred method of attachment is by covalent means. Following
 attachment of a specific binding member, the surface may be further
 treated with materials such as serum, proteins, or other blocking agents
 to minimize non-specific binding. The surface also may be scanned either
 at the site of manufacture or point of use to verify its suitability for
 assay purposes. The scanning process is not anticipated to alter the
 specific binding properties of the test piece.
 While the present invention discloses the preference for the use of solid
 phases, it is contemplated that the reagents such as antibodies, proteins
 and peptides of the present invention can be utilized in non-solid phase
 assay systems. These assay systems are known to those skilled in the art,
 and are considered to be within the scope of the present invention.
 It is contemplated that the reagent employed for the assay can be provided
 in the form of a test kit with one or more containers such as vials or
 bottles, with each container containing a separate reagent such as a
 probe, primer, monoclonal antibody or a cocktail of monoclonal antibodies,
 or a polypeptide (e.g. recombinantly, synthetically produced or purified)
 employed in the assay. The polypeptide is selected from the group
 consisting of those encoded by markers represented by SEQUENCE ID NOS
 1-13, and fragments thereof. Other components such as buffers, controls
 and the like, known to those of ordinary skill in art, may be included in
 such test kits. It also is contemplated to provide test kits which have
 means for collecting test samples comprising accessible body fluids, e.g.,
 blood, urine, saliva and stool. Such tools useful for collection
 ("collection materials") include lancets and absorbent paper or cloth for
 collecting and stabilizing blood; swabs for collecting and stabilizing
 saliva; cups for collecting and stabilizing urine or stool samples.
 Collection materials, papers, cloths, swabs, cups and the like, may
 optionally be treated to avoid denaturation or irreversible adsorption of
 the sample. The collection materials also may be treated with or contain
 preservatives, stabilizers or antimicrobial agents to help maintain the
 integrity of the specimens. Test kits designed for the collection,
 stabilization and preservation of test specimens obtained by surgery or
 needle biopsy are also useful. It is contemplated that all kits may be
 configured in two components which can be provided separately; one
 component for collection and transport of the specimen and the other
 component for the analysis of the specimen. The collection component, for
 example, can be provided to the open market user while the components for
 analysis can be provided to others such as laboratory personnel for
 determination of the presence, absence or amount of analyte. Further, kits
 for the collection, stabilization and preservation of test specimens may
 be configured for use by untrained personnel and may be available in the
 open market for use at home with subsequent transportation to a laboratory
 for analysis of the test sample.
 In Vivo Antibody Use.
 Antibodies of the present invention can be used in vivo; that is, they can
 be injected into patients suspected of having diseases of the urinary
 tract for diagnostic or therapeutic uses. The use of antibodies for in
 vivo diagnosis is well known in the art. Sumerdon et al., Nucl. Med. Biol
 17:247-254 (1990) have described an optimized antibody-chelator for the
 radioimmunoscintographic imaging of carcinoembryonic antigen (CEA)
 expressing tumors using Indium-111 as the label. Griffin et al., J Clin
 Onc 9:631-640 (1991) have described the use of this agent in detecting
 tumors in patients suspected of having recurrent colorectal cancer. The
 use of similar agents with paramagnetic ions as labels for magnetic
 resonance imaging is known in the art (R. B. Lauffer, Magnetic Resonance
 in Medicine 22:339-342 (1991). It is anticipated that antibodies directed
 against antigen encoded by markers represented by SEQUENCE ID NOS 1-13 can
 be injected into patients suspected of having a disease of the urinary
 tract such as bladder cancer for the purpose of diagnosing or staging the
 disease status of the patient. The label used will depend on the imaging
 modality chosen. Radioactive labels such as Indium-111, Technetium-99m, or
 Iodine-131 can be used for planar scans or single photon emission computed
 tomography (SPECT). Positron emitting labels such as Fluorine-19 can also
 be used for positron emission tomography (PET). For MRI, paramagnetic ions
 such as Gadolinium (III) or Manganese (II) can be used. Localization of
 the label within the urinary tract or external to the urinary tract may
 allow determination of spread of the disease. The amount of label within
 the urinary tract may allow determination of the presence or absence of
 cancer of the urinary tract.
 For patients known to have a disease of the urinary tract, injection of an
 antibody directed against antigen encoded by markers represented by
 SEQUENCE ID NOS 1-13 may have therapeutic benefit. The antibody may exert
 its effect without the use of attached agents by binding to antigen
 encoded by markers represented by SEQUENCE ID NOS 1-13 expressed on or in
 the tissue or organ. Alternatively, the antibody may be conjugated to
 cytotoxic agents such as drugs, toxins, or radionuclides to enhance its
 therapeutic effect. Garnett and Baldwin, Cancer Research 46:2407-2412
 (1986) have described the preparation of a drug-monoclonal antibody
 conjugate. Pastan et al., Cell 47:641-648 (1986) have reviewed the use of
 toxins conjugated to monoclonal antibodies for the therapy of various
 cancers. Goodwin and Meares, Cancer Supplement 80:2675-2680 (1997) have
 described the use of Yittrium-90 labelled monoclonal antibodies in various
 strategies to maximize the dose to tumor while limiting normal tissue
 toxicity. Other known cytotoxic radionuclides include Copper-67,
 Iodine-131, and Rhenium-186 all of which can be used to label monoclonal
 antibodies directed against antigen encoded by markers represented by
 SEQUENCE ID NOS 1-13 for the treatment of cancer of the bladder.
 The present invention will now be described by way of examples, which are
 meant to illustrate, but not to limit, the scope of the present invention.
 EXAMPLES
 Example 1
 Identification of Bladder Tumor Library EST Clones
 Experiments performed in support of the present invention suggested that a
 multiplicity of cytokeratins may be up-regulated in bladder tumors. A
 search was conducted in Genbank for title entries with the keywords
 keratin or cytokeratin. This search resulted in a collection of 57 Genbank
 entries which were annotated in the LifeSeq.TM. database. The number of
 EST's occurring in tumor, normal and other libraries, categorized by
 keratin or cytokeratin type, were classified according to type utilizing
 all available published information from literature and sequences in
 GenBank. Because the cytokeratin type designated 50 kDa was not obviously
 categorized it was treated as a separate class.
 The above search data are summarized in Table 1, which further includes the
 ratios of the numbers of EST's in "bladder tumor" to "bladder normal"
 libraries. The table has been sorted according to this ratio with the
 highest ratio at the top of the table. In the case where there are no
 occurrences in the normal libraries, the ratio is expressed as greater
 than (&gt;) a minimal value, rather than infinity.
 TABLE 1
 Number of Clones in Libraries
 Keratin/ Bladder
 cyto- Tumor to
 keratin Normal Sequence Genbank
 type Normal Tumor Ratio Other ID No. ID No.
 50 kDa 1 31 31 231 1 187604
 5 0 27 &gt;27 550 2 186697
 16 0 20 &gt;20 120 3 186676
 17 3 53 18 736 4 30378
 6 2 31 16 337 5 consen-
 sus*
 18 1 15 15 785 6 34036
 7 2 26 13 275 7 186729
 8 5 25 5 839 8 191399
 19 8 31 4 570 9 184568
 13 6 15 3 83 10 30376
 15 0 3 &gt;3 144 11 34070
 14 0 1 &gt;1 85
 10 0 1 &gt;1 18
 20 0 0 115
 4 2 0 16
 3 0 0 1
 2 0 0 14
 Hair type 0 1 3
 1
 *consensus sequence derived from keratin/cytokeratin type 6a, 6c, 6e, and
 6f, respectively, Genbank Index Nos. 908769, 186699, 908602, and 908804.
 In Table 1, the number of clones in each of the libraries are summed for
 each keratin or cytokeratin type. Sequence ID No. refers to the prototypic
 sequence that is presented in the Sequence Listing of the present
 application (the corresponding Genbank ID No. is the source of the
 sequence). In the case of cytokeratin type 6, due to the published variant
 forms, a consensus sequence was derived for Seq ID No. 5, based on the
 individual sequences as shown in the footnote to Table 1.
 Representative sequences corresponding to the keratin or cytokeratin types
 are presented as the SEQUENCE ID Nos. listed herein, and, further, each
 representative sequence is referenced to a Genbank Index number. Together,
 these data permit the selection of those sequences that are highly
 expressed in different bladder tumors compared with the normal state.
 These representative sequences can be used to define translation open
 reading frames by standard methods. Further, analysis of the protein
 structure can be used to define epitopes that will be unique for the
 preferentially expressed species and minimize cross-reactivity with the
 non- or low-expressed species.
 Example 2
 Cas Gene Expression in Bladder Tumor Libraries
 A cellular apoptosis susceptibility (CAS) gene (PCT application WO 9640713)
 was found to be represented in EST libraries from bladder tumors but not
 in libraries derived from normal bladders. Table 2 presents the
 occurrences of clones with sequences matching CAS mRNA in the LifeSeqTm
 database.
 TABLE 2
 Normal Tumor
 Represented in Represented in
 No. of number of No. of number of
 Tissue Type Clones libraries Clones libraries
 Bladder 0 0 5 5
 Brain 11 7 9 5
 Breast 5 4 8 5
 Colon 3 3 2 2
 Lung 5 4 8 5
 Prostate 4 3 1 1
 Total Others 73
 While this gene is expressed in libraries from a variety of normal and
 tumor tissues, it can be seen that it is present in 5 independent bladder
 tumor libraries but it is not present in normal libraries. The expression
 in bladder tumor indicates that the sequence provides useful probes for
 bladder tumors, for example, when test urine is probed for the presence of
 (1) an mRNA having sequences complementary to SEQUENCE ID NO. 12, or (2) a
 protein, or fragments thereof, encoded by the sequence.
 Example 3
 Mat-8 Gene Expression in Bladder Tumor Libraries
 A chloride channel protein found in human breast cancer cells has been
 designated mat-8 (mammary tumor 8 kD: PCT application WO 96/05322,
 Morrison and Leder). Clones in the LifeSeq.TM. database carrying the
 sequence coding for this protein were investigated--the results of an
 analysis similar to that of Example 2 are presented in Table 3.
 TABLE 3
 Normal Tumor
 Represented in Represented in
 No. of number of No. of number of
 Tissue Type Clones libraries Clones libraries
 Bladder 0 0 7 4
 Breast 6 3 10 3
 Colon 56 12 5 2
 Lung 12 6 1 1
 Prostate 29 10 4 2
 Total Others 37
 The gene appears to be expressed in a variety of organs and tissues, but 7
 clones were found in 5 bladder tumor libraries and none from any normal
 bladder. The expression in bladder tumor indicates that the sequence
 provides useful probes for bladder tumors, for example, when test urine is
 probed for the presence of (1) an mRNA having sequences complementary to
 SEQUENCE ID NO. 13, or (2) a protein, or fragments thereof, encoded by the
 sequence.
 It is also noted that this gene appears to be down-regulated in tumors in
 certain organs: 98 occurrences in normal colon compared with 10 in colon
 tumors; 41 occurrences in normal prostates compared with 7 in prostate
 tumors.
 Example 4
 Nucleic Acid Preparation
 A. RNA Extraction from Tissue.
 Total RNA is isolated from urinary tract tissues and from non-urinary tract
 tissues. Various methods are utilized, including but not limited to the
 lithium chloride/urea technique, known in the art and described by Kato et
 al., (J. Virol. 61:2182-2191, 1987), and TRIZOlTM (Gibco-BRL, Grand
 Island, N.Y.).
 Briefly, tissue is placed in a sterile conical tube on ice and 10-15
 volumes of 3 M LiCl, 6 M urea, 5 mM EDTA, 0.1 M .beta.-mercaptoethanol, 50
 mM Tris-HCl (pH 7.5) are added. The tissue is homogenized with a
 Polytron.RTM. homogenizer (Brinkman Instruments, Inc., Westbury, N.Y.) for
 30-50 sec on ice. The solution is transferred to a 15 ml plastic
 centrifuge tube and placed overnight at -20.degree. C. The tube is
 centrifuged for 90 min at 9,000.times.g at 0-4.degree. C. and the
 supernatant is immediately decanted. Ten ml of 3 M LiCi are added and the
 tube is vortexed for 5 sec. The tube is centrifuged for 45 min at
 11,000.times.g at 0-4.degree. C. The decanting, resuspension in LiCl, and
 centrifugation is repeated and the final pellet is air dried and suspended
 in 2 ml of 1 mM EDTA, 0.5% SDS, 10 mM Tris (pH 7.5). Twenty microliters
 (20 .mu.l) of Proteinase K (20 mg/ml) are added, and the solution is
 incubated for 30 min at 37.degree. C. with occasional mixing. One-tenth
 volume (0.22-0.25 ml) of 3 M NaCl is added and the solution is vortexed
 before transfer into another tube containing 2 ml of
 phenol/chloroform/isoamyl alcohol (PCI). The tube is vortexed for 1-3 sec
 and centrifuged for 20 min at 3,000.times.g at 10.degree. C. The PCI
 extraction is repeated and followed by two similar extractions with
 chloroform/isoamyl alcohol (CI). The final aqueous solution is transferred
 to a chilled 15 ml Corex glass tube containing 6 ml of absolute ethanol,
 the tube is covered with parafilm, and placed at -20.degree. C. overnight.
 The tube is centrifuged for 30 min at 10,000.times.g at 0-4.degree. C. and
 the ethanol supernatant is decanted immediately. The RNA pellet is washed
 four times with 10 ml of 75% ice-cold ethanol and the final pellet is air
 dried for 15 min at room temperature. The RNA is suspended in 0.5 ml of 10
 mM TE (pH 7.6, 1 mM EDTA) and its concentration is determined
 spectrophotometrically. RNA samples are aliquoted and stored at
 -70.degree. C. as ethanol precipitates.
 The quality of the RNA is determined by agarose gel electrophoresis (see
 Example 6, Northern Blot Analysis) and staining with 0.5 .mu.g/ml ethidium
 bromide for one hour. RNA samples that do not contain intact rRNAs are
 excluded from the study.
 Alternatively, for RT-PCR analysis, 1 ml of Ultraspec RNA reagent is added
 to 120 mg of pulverized tissue in a 2.0 ml polypropylene microfuge tube,
 homogenized with a Polytron.RTM. homogenizer (Brinkman Instruments, Inc.,
 Westbury, N.Y.) for 50 sec and placed on ice for 5 min. Then, 0.2 ml of
 chloroform is added to each sample, followed by vortexing for 15 sec. The
 sample is placed on ice for another 5 min, followed by centrifugation at
 12,000.times.g for 15 min at 4.degree. C. The upper layer is collected and
 transferred to another RNase-free 2.0 ml microfuge tube. An equal volume
 of isopropanol is added to each sample, and the solution is placed on ice
 for 10 min. The sample is centrifuged at 12,000.times.g for 10 min at
 4.degree. C., and the supernatant is discarded. The remaining pellet is
 washed twice with cold 75% ethanol, resuspended by vortexing, and the
 resuspended material is then pelleted by centrifugation at 7500.times.g
 for 5 min at 4.degree. C. Finally, the RNA pellet is dried in a Speedvac
 (Savant, Farmingdale, N.Y.) for 5 min and reconstituted in RNase-free
 water.
 B. RNA Extraction from Blood Mononuclear Cells.
 Mononuclear cells are isolated from blood samples from patients by
 centrifugation using Ficoll-Hypaque as follows. A 10 ml volume of whole
 blood is mixed with an equal volume of RPMI Medium (Gibco-BRL, Grand
 Island, N.Y.). This mixture is then underlayed with 10 ml of
 Ficoll-Hypaque (Pharmacia, Piscataway, N.J.) and centrifuged for 30
 minutes at 200.times.g. The buffy coat containing the mononuclear cells is
 removed, diluted to 50 ml with Dulbecco's PBS (Gibco-BRL, Grand Island,
 N.Y.) and the mixture centrifuged for 10 minutes at 200.times.g. After two
 washes, the resulting pellet is resuspended in Dulbecco's PBS to a final
 volume of 1 ml.
 RNA is prepared from the isolated mononuclear cells as described by N. Kato
 et al., J. Virology 61: 2182-2191 (1987). Briefly, the pelleted
 mononuclear cells are brought to a final volume of 1 ml and then are
 resuspended in 250 .mu.L of PBS and mixed with 2.5 ml of 3M LiCl, 6M urea,
 5mM EDTA, 0.1M 2-mercaptoethanol, 50 mM Tris-HCl (pH 7.5). The resulting
 mixture is homogenized and incubated at -20.degree. C. overnight. The
 homogenate is centrifuged at 8,000 RPM in a Beckman J2-21M rotor for 90
 minutes at 0-4.degree. C. The pellet is resuspended in 10 ml of 3M LiCl by
 vortexing and then centrifuged at 10,000 RPM in a Beckman J2-21M rotor
 centrifuge for 45 minutes at 0-4.degree. C. The resuspending and pelleting
 steps then are repeated. The pellet is resuspended in 2 ml of 1 mM EDTA,
 0.5% SDS, 10 mM Tris (pH 7.5) and 400 gg Proteinase K with vortexing and
 then it is incubated at 37.degree. C. for 30 minutes with shaking. One
 tenth volume of 3M NaCl then is added and the mixture is vortexed.
 Proteins are removed by two cycles of extraction with
 phenol/chloroform/isoamyl alcohol (PCI) followed by one extraction with
 chloroform/isoamyl alcohol (CI). RNA is precipitated by the addition of 6
 ml of absolute ethanol followed by overnight incubation at -20.degree. C.
 After the precipitated RNA is collected by centrifugation, the pellet is
 washed 4 times in 75% ethanol. The pelleted RNA is then dissolved in
 solution containing 1 mM EDTA, 10 mM Tris-HCl (pH 7.5).
 Non-urinary tract tissues are used as negative controls. The mRNA can be
 further purified from total RNA by using commercially available kits such
 as oligo dT cellulose spin columns (RediCol.TM. from Pharmacia, Uppsala,
 Sweden) for the isolation of poly-adenylated RNA. Total RNA or mRNA can be
 dissolved in lysis buffer (SM guanidine thiocyanate, 0.1 M EDTA, pH 7.0)
 for analysis in the ribonuclease protection assay.
 C. RNA Extraction from polysomes.
 Tissue is minced in saline at 4.degree. C. and mixed with 2.5 volumes of
 0.8 M sucrose in a TK.sub.150 M (150 mM KCl, 5 mM MgCl.sub.2, 50 mM
 Tris-HCl, pH 7.4) solution containing 6 mM 2-mercaptoethanol. The tissue
 is homogenized in a Teflon-glass Potter homogenizer with five strokes at
 100-200 rpm followed by six strokes in a Dounce homogenizer, as described
 by B. Mechler, Methods in Enzymology 152:241-248 (1987). The homogenate
 then is centrifuged at 12,000.times.g for 15 min at 4.degree. C. to
 sediment the nuclei. The polysomes are isolated by mixing 2 ml of the
 supernatant with 6 ml of 2.5 M sucrose in TK.sub.150 M and layering this
 mixture over 4 ml of 2.5 M sucrose in TK.sub.150 M in a 38 ml polyallomer
 tube. Two additional sucrose TK.sub.150 M solutions are successively
 layered onto the extract fraction; a first layer of 13 ml 2.05 M sucrose
 followed by a second layer of 6 ml of 1.3 M sucrose. The polysomes are
 isolated by centrifuging the gradient at 90,000.times.g for 5 hr at
 4.degree. C. The fraction then is taken from the 1.3 M sucrose/2.05 M
 sucrose interface with a siliconized pasteur pipette and diluted in an
 equal volume of TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA). An equal volume of
 90.degree. C. SDS buffer (1% SDS, 200 mM NaCl, 20 mM Tris-HCl, pH 7.4) is
 added and the solution is incubated in a boiling water bath for 2 min.
 Proteins next are digested with a Proteinase K digestion (50 mg/ml) for 15
 min at 37.degree. C. The mRNA is purified with 3 equal volumes of
 phenol-chloroform extractions followed by precipitation with 0.1 volume of
 2 M sodium acetate (pH 5.2) and 2 volumes of 100% ethanol at -20.degree.
 C. overnight. The precipitated RNA is recovered by centrifugation at
 12,000.times.g for 10 min at 4.degree. C. The RNA is dried and resuspended
 in TE (pH 7.4) or distilled water. The resuspended RNA then can be used in
 a slot blot or dot blot hybridization assay to check for the presence of
 cytokeratin mRNA (see Example 6).
 The quality of nucleic acid and proteins is dependent on the method of
 preparation used. Each sample may require a different preparation
 technique to maximize isolation efficiency of the target molecule. These
 preparation techniques are within the skill of the ordinary artisan.
 Example 5
 Ribonuclease Protection Assay
 A. Synthesis of Labeled Complementary RNA (cRNA) Hybridization.
 Probe and Unlabeled Sense Strand.
 Labeled antisense and unlabeled sense riboprobes are transcribed from the
 cytokeratin gene cDNA sequence which contains a 5' RNA polymerase promoter
 such as SP6 or T7. The sequence may be from a vector containing the
 appropriate cytokeratin cDNA insert, or from a PCR-generated product of
 the insert using PCR primers which incorporate a 5' RNA polymerase
 promoter sequence. For example, the described plasmid, clone 1890382 or
 another comparable clone, containing the cytokeratin gene cDNA sequence,
 flanked by opposed SP6 and T7 or other RNA polymerase promoters, is
 purified using a Qiagen Plasmid Purification Kit (Qiagen, Chatsworth,
 Calif.). Then 10 .mu.g of the plasmid DNA are linearized by cutting with
 an appropriate restriction enzyme such as Dde I for 1 hr at 37.degree. C.
 The linearized plasmid DNA is purified using the QlAprep Kit (Qiagen,
 Chatsworth, Calif.) and used for the synthesis of antisense transcript
 from the appropriate promoter using the Riboprobe.RTM. in vitro
 Transcription System (Promega Corporation, Madison, Wis.), as described by
 the supplier's instructions, incorporating either (alpha.sup.32 P) CTP
 (Amersham Life Sciences, Inc. Arlington Heights, Ill.) or biotinylated CTP
 as a label. To generate the sense strand, 10 .mu.g of the purified plasmid
 DNA are cut with restriction enzymes, such as Xba I and Not I, and
 transcribed as above from the appropriate promoter. Both sense and
 antisense strands are isolated by spin column chromatography. Unlabeled
 sense strand is quantitated by UV absorption at 260 nm.
 B. Hybridization of Labeled Probe to Target.
 Frozen tissue is pulverized to powder under liquid nitrogen and 100-500 mg
 are dissolved in 1 ml of lysis buffer, available as a component of the
 Direct Protect.TM. Lysate RNase Protection Kit (Ambion, Inc., Austin,
 Tex.). Further dissolution can be achieved using a tissue homogenizer. In
 addition, a dilution series of a known amount of sense strand in mouse
 liver lysate is made for use as a positive control. Finally, 45 .mu.l of
 solubilized tissue or diluted sense strand are mixed directly with either;
 1) 1.times.10.sup.5 cpm of radioactively labeled probe, or 2) 250 pg of
 non-isotopically labeled probe in 5 .mu.l of lysis buffer. Hybridization
 is allowed to proceed overnight at 37.degree. C. See, T. Kaabache et al.,
 Anal. Biochem. 232:225-230 (1995).
 C. RNase Digestion.
 RNA that is not hybridized to probe is removed from the reaction as per the
 Direct Protect.TM. protocol using a solution of RNase A and RNase T1 for
 30 min at 37.degree. C., followed by removal of RNase by Proteinase K
 digestion in the presence of sodium sarcosyl. Hybridized fragments
 protected from digestion are then precipitated by the addition of an equal
 volume of isopropanol and placed at -70.degree. C. for 3 hr. The
 precipitates are collected by centrifugation at 12,000.times.g for 20 min.
 D. Fragment Analysis.
 The precipitates are dissolved in denaturing gel loading dye (80%
 formamide, 10 mM EDTA (pH 8.0), 1 mg/ml xylene cyanol, 1 mg/ml bromophenol
 blue), heat denatured, and electrophoresed in 6% polyacrylamide TBE, 8 M
 urea denaturing gels. The gels are imaged and analyzed using the STORM.TM.
 storage phosphor autoradiography system (Molecular Dynamics, Sunnyvale,
 Calif.). Quantitation of protected fragment bands, expressed in femtograms
 (fg), is achieved by comparing the peak areas obtained from the test
 samples to those from the known dilutions of the positive control sense
 strand (see Section B, supra). The results are expressed in molecules of
 cytokeratin RNA/cell and as a image rating score. In cases where
 non-isotopic labels are used, hybrids are transferred from the gels to
 membranes (nylon or nitrocellulose) by blotting and then analyzed using
 detection systems that employ streptavidin alkaline phosphatase conjugates
 and chemiluminesence or chemifluoresence reagents.
 Detection of a product in urinary tract tissue, in urine, or in cells
 exfoliated into urine comprising a sequence selected from the group
 consisting of SEQUENCE ID NOS 1-13, and fragments or complements thereof
 suggests a diagnosis of urinary tract cancer.
 Example 6
 Northern Blotting
 The Northern blot technique is used to identify a specific size RNA
 fragment from a complex population of RNA using gel electrophoresis and
 nucleic acid hybridization. Northern blotting is well-known technique in
 the art. Briefly, 5-10 .mu.g of total RNA (see Example 3) are incubated in
 15 .mu.l of a solution containing 40 mM morphilinopropanesulfonic acid
 (MOPS) (pH 7.0), 10 mM sodium acetate, 1 mM EDTA, 2.2 M formaldehyde, 50%
 v/v formamide for 15 min at 65.degree. C. The denatured RNA is mixed with
 2 .mu.l of loading buffer (50% glycerol, 1 mM EDTA, 0.4% bromophenol blue,
 0.4% xylene cyanol) and loaded into a denaturing 1.0% agarose gel
 containing 40 mM MOPS (pH 7.0), 10 mM sodium acetate, 1 mM EDTA and 2.2 M
 formaldehyde. The gel is electrophoresed at 60 V for 1.5 hr and rinsed in
 RNAse free water. RNA is transferred from the gel onto nylon membranes
 (Brightstar-Plus, Ambion, Inc., Austin, Tex.) for 1.5 hours using the
 downward alkaline capillary transfer method (Chomczynski, Anal. Biochem.
 201:134-139, 1992). The filter is rinsed with IX SSC, and RNA is
 crosslinked to the filter using a Stratalinkerim (Stratagene, Inc., La
 Jolla, Calif.) on the autocrosslinking mode and dried for 15 min. The
 membrane is then placed into a hybridization tube containing 20 ml of
 preheated prehybridization solution (5X SSC, 50% formamide, 5X Denhardt's
 solution, 100 .mu.g/ml denatured salmon sperm DNA) and incubated in a
 42.degree. C. hybridization oven for at least 3 hr. While the blot is
 prehybridizing, a .sup.32 P-labeled random-primed probe is generated using
 the CYTOKERATIN insert fragment (obtained, for example, by digesting clone
 1890382 with XbaI and NotI, or another comparable clone with appropriate
 restriction enzymes) using Random Primer DNA Labeling System (Life
 Technologies, Inc., Gaithersburg, Md.) according to the manufacturer's
 instructions. Half of the probe is boiled for 10 min, quick chilled on ice
 and added to the hybridization tube. Hybridization is performed at
 42.degree. C. for at least 12 hr. The hybridization solution is discarded
 and the filter is washed in 30 ml of 3X SSC, 0. 1% SDS at 42.degree. C.
 for 15 min, followed by 30 ml of 3X SSC, 0.1% SDS at 42.degree. C. for 15
 min. The filter is wrapped in Saran Wrap, exposed to Kodak XAR-Omat film
 for 8-96 hr, and the film is developed for analysis. Detection of a
 product in urinary tract tissue, in urine, or in cells exfoliated into
 urine comprising a sequence selected from the group consisting of SEQUENCE
 ID NOS 1-13, and fragments or complements thereof suggests a diagnosis of
 urinary tract cancer.
 Example 7
 Blot/Slot Blot
 Dot and slot blot assays are quick methods to evaluate the presence of a
 specific nucleic acid sequence in a complex mix of nucleic acid. To
 perform such assays, up to 50 jig of RNA are mixed in 50 Il of 50%
 formamide, 7% formaldehyde, 1X SSC, incubated 15 min at 68.degree. C., and
 then cooled on ice. Then, 100 Al of 20X SSC are added to the RNA mixture
 and loaded under vacuum onto a manifold apparatus that has a prepared
 nitrocellulose or nylon membrane. The membrane is soaked in water, 20X SSC
 for 1 hour, placed on two sheets of 20X SSC prewet Whatman #3 Dot filter
 paper, and loaded into a slot blot or dot blot vacuum manifold apparatus.
 The slot blot is analyzed with probes prepared and labeled as described in
 Example 4, supra. Detection of a product in urinary tract tissue, in
 urine, or in cells exfoliated into urine comprising a sequence selected
 from the group consisting of SEQUENCE ID NOS 1-13, and fragments or
 complements thereof suggests a diagnosis of urinary tract cancer.
 Other methods and buffers which can be utilized in the methods described in
 Examples 5, 6, and 7 but not specifically detailed herein, are known in
 the art and are described in J. Sambrook et al., supra, which is
 incorporated herein by reference.
 Example 8
 In Situ Hybridization
 This method is useful to directly detect specific target nucleic acid
 sequences in cells using detectable nucleic acid hybridization probes.
 Tissues are prepared with cross-linking fixative agents such as
 paraformaldehyde or glutaraldehyde for maximum cellular RNA retention.
 See, L. Angerer et al., Methods in Cell Biol. 35:37-71 (1991). Briefly,
 the tissue is placed in greater than 5 volumes of 1% glutaraldehyde in 50
 mM sodium phosphate, pH 7.5 at 4.degree. C. for 30 min. The solution is
 changed with fresh glutaraldehyde solution (1% glutaraldehyde in 50 mM
 sodium phosphate, pH 7.5) for a further 30 min fixing. The fixing solution
 should have an osmolality of approximately 0.375% NaCl. The tissue is
 washed once in isotonic NaCl to remove the phosphate.
 The fixed tissues then are embedded in paraffin as follows. The tissue is
 dehydrated though a series of increasing ethanol concentrations for 15 min
 each: 50% (twice), 70% (twice), 85%, 90% and then 100% (twice). Next, the
 tissue is soaked in two changes of xylene for 20 min each at room
 temperature. The tissue is then soaked in two changes of a 1:1 mixture of
 xylene and paraffin for 20 min each at 60.degree. C.; and then in three
 final changes of paraffin for 15 min each.
 Next, the tissue is cut in 5 .mu.m sections using a standard microtome and
 placed on a slide previously treated with a tissue adhesive such as
 3-aminopropyltriethoxysilane.
 Paraffin is removed from the tissue by two 10 min xylene soaks and
 rehydrated in a series of decreasing ethanol concentrations: 99% twice,
 95%, 85%, 70%, 50%, 30%, and then distilled water twice. The sections are
 pre-treated with 0.2 M HCl for 10 min and permeabilized with 2 .mu.g/ml
 Proteinase K at 37.degree. C. for 15 min.
 Labeled riboprobes transcribed from the plasmid (see Example 4) are
 hybridized to the prepared tissue sections and incubated overnight at
 56.degree. C. in 3X standard saline extract and 50% formamide. Excess
 probe is removed by washing in 2X standard saline citrate and 50%
 formamide followed by digestion with 100 .mu.g/ml RNase A at 37.degree. C.
 for 30 min. Fluorescence probe is visualized by illumination with
 ultraviolet (UV) light under a microscope. Fluorescence in the cytoplasm
 is indicative of specific mRNA. Alternatively, the sections can be
 visualized by autoradiography. Detection of a product in urinary tract
 tissue, in urine, or in cells exfoliated into urine comprising a sequence
 selected from the group consisting of SEQUENCE ID NOS 1-13, and fragments
 or complements thereof suggests a diagnosis of urinary tract cancer.
 Example 9
 Reverse Transcription PCR
 A. One Step RT-PCR Assay.
 Target-specific primers are designed to detect the above-described target
 sequences by reverse transcription PCR using methods known in the art. One
 step RT-PCR is a sequential procedure that performs both RT and PCR in a
 single reaction mixture. The procedure is performed in a 200 .mu.l
 reaction mixture containing 50 mM (N,N,-bis[2-Hydroxyethyl]glycine), pH
 8.15, 81.7 mM KOAc, 33.33 mM KOH, 0.01 mg/ml bovine serum albumin, 0.1 mM
 ethylene diaminetetraacetic acid, 0.02 mg/ml NaN3, 8% w/v glycerol, 150
 .mu.M each of dNTP, 0.25 .mu.M each primer, 5U rTth polymerase, 3.25 mM
 Mn(OAc).sub.2 and 5 .mu.l of target RNA (see Example 3). Since RNA and the
 rTth polymerase enzyme are unstable in the presence of Mn(OAc).sub.2, the
 Mn(OAc).sub.2 should be added just before target addition. Optimal
 conditions for cDNA synthesis and thermal cycling readily can be
 determined by those skilled in the art. The reaction is incubated in a
 Perkin-Elmer Thermal Cycler 480. Conditions which may be found useful
 include cDNA synthesis at 60.degree.-70.degree. C. for 15-45 min and 30-45
 amplification cycles at 94.degree. C., 1 min; 55.degree.-70.degree. C., 1
 min; 72.degree. C., 2 min. One step RT-PCR also may be performed by using
 a dual enzyme procedure with Taq polymerase and a reverse transcriptase
 enzyme, such as MMLV (Moloney murine leukemia virus) or AMV (avian
 myeloblastosis virus) RT (reverse transcriptase) enzymes.
 B. Traditional RT-PCR.
 Cytokeratin genes share many homologous regions. Therefore, care is
 required in the design of RT-PCR primers to ensure the desired
 specificity. For example, to guide the primer design, all 100 bp strings
 of the cytokeratin ck5 mRNA sequence (SEQUENCE ID NO 2) were compared with
 the sequences of all other available cytokeratin mRNAs. A ck5 substring
 was flagged as a possible region for a primer provided that it was no more
 than 70% identical to any other 100 bp substring in any other cytokeratin.
 Then, using primer analysis software, for example, Oligo.TM. (Version
 4.0), RT-PCR primers (SEQUENCE ID NO 14 and SEQUENCE ID NO 15) were
 designed. A traditional two-step RT-PCR reaction was performed, as
 described by K. Q. Hu et al., Virology 181:721-726 (1991). Briefly, 0.5
 .mu.g of extracted mRNA (see Example 3) was reverse transcribed in a 20
 .mu.l reaction mixture containing 1X PCR II buffer (Perkin-Elmer), 5 mM
 MgCl2, 1 mM each dNTP, 20 U RNasin, 2.5 .mu.M random hexamers, and 50 U
 MMLV RT. Reverse transcription was performed at room temperature for 10
 min, 42.degree. C. for 30 min in a PE480 thermal cycler (Perkin-Elmer),
 followed by further incubation at 95.degree. C. for 5 min to inactivate
 the RT. PCR was performed using 2 .mu.l of the cDNA reaction in a final
 PCR reaction volume of 50 tl containing 1X PCR II buffer (Perkin-Elmer),
 50 mM KCl, 1.5 mM MgC12, 200 gM dNTPs, 0.5 .mu.M of each sense and
 antisense primer, (SEQUENCE ID NO 14) and (SEQUENCE ID NO 15),
 respectively, and 2.5 U of Taq polymerase. The reaction was incubated in a
 MJ Research Model PTC-200, as follows: 35 cycles of amplification
 (94.degree. C., 45 sec; 62.degree. C., 45 sec; 72.degree. C., 2 min.); a
 final extension (72.degree. C., 5 min); and a soak at 4.degree. C.
 C. PCR Fragment Analysis.
 The correct products were verified by size determination using gel
 electrophoresis with a SYBR.RTM. Green I nucleic acid gel stain (Molecular
 Probes, Eugene, Oreg.). Gels were stained with SYBR.RTM. Green I at a
 1:10,000 dilution in 1X TBE for 45 min. The gel, FIG. 1, was imaged using
 a STORM.TM. imaging system (Molecular Dynamics, Sunnyvale, Calif.). In
 FIG. 1, the lane contents were as follows: lane 1, 100 bp-ladder molecular
 weight markers; lane 2, placental DNA; lane 3, bladder cancer RNA; lane 4,
 normal bladder RNA; lane 5, normal bladder RNA; lane 6, normal bladder
 RNA; lane 7, bladder cancer RNA; lane 8, prostate BPH RNA; lane 9, normal
 colon RNA; lane 10, breast cancer RNA; and lane 11, colon cancer RNA. FIG.
 1 shows a 607 bp cytokeratin ck5-specific PCR amplification product in
 lanes 3, 7, 8 and 9, indicating that cytokeratin ck5 mRNA was present in 2
 of 2 bladder cancer samples and 0 of 3 normal bladder samples tested. In
 FIG. 1, the 607 bp cytokeratin ck5-specific PCR amplification product is
 also observed (faintly) in one normal colon sample (lane 9), and in one
 prostate BPH sample (lane 8). These data suggest that cytokeratin ck5 mRNA
 expression is bladder cancer specific. Therefore, detection of a product
 in urinary tract tissue, in urine, or in cells exfoliated into urine
 comprising SEQUENCE ID NO 2, and fragments or complements thereof
 indicates the presence of ck5 mRNA suggesting a diagnosis of urinary tract
 cancer.
 Similarly, detection of a product in urinary tract tissue, in urine, or in
 cells exfoliated into urine comprising a sequence selected from the group
 consisting of SEQUENCE ID NO 1 and SEQUENCE ID NOS 3-13, and fragments or
 complements thereof suggests a diagnosis of urinary tract cancer.
 Example 10
 OH-PCR
 A. Probe selection and Labeling.
 Target-specific primers and probes are designed to detect the
 above-described target sequences by oligonucleotide hybridization PCR.
 International Publication Nos WO 92/10505, published June 25, 1992, and WO
 92/11388, published Jul. 9, 1992, teach methods for labeling
 oligonucleotides at their 5' and 3' ends, respectively. According to one
 known method for labeling an oligonucleotide, a label-phosphoramidite
 reagent is prepared and used to add the label to the oligonucleotide
 during its synthesis. For example, see N. T. Thuong et al., Tet. Letters
 29(46):5905-5908 (1988); or J. S. Cohen et al., published U.S. patent
 application Ser. No. 07/246,688 (NTIS ORDER No. PAT-APPL-7-246,688)
 (1989). Preferably, probes are labeled at their 3' end to prevent
 participation in PCR and the formation of undesired extension products.
 For one step OH-PCR, the probe should have a TM at least 15.degree. C.
 below the T.sub.M of the primers. The primers and probes are utilized as
 specific binding members, with or without detectable labels, using
 standard phosphoramidite chemistry and/or post-synthetic labeling methods
 which are well-known to one skilled in the art.
 B. One Step Oligo Hybridization PCR.
 OH-PCR is performed on a 200 .mu.l reaction containing 50 mM
 (N,N,-bis[2-Hydroxyethyl]glycine), pH 8.15, 81.7 mM KOAc, 33.33 mM KOH,
 0.01 mg/ml bovine serum albumin, 0.1 mM ethylene diaminetetraacetic acid,
 0.02 mg/ml NaN.sub.3, 8% w/v glycerol, 150 .mu.M each of dNTP, 0.25 .mu.M
 each primer, 3.75 nM probe, 5U rTth polymerase, 3.25 mM Mn(OAc).sub.2 and
 5 .mu.l blood equivalents of target (see Example 3). Since RNA and the
 rTth polymerase enzyme are unstable in the presence of Mn(OAc).sub.2, the
 Mn(OAc).sub.2 should be added just before target addition. The reaction is
 incubated in a Perkin-Elmer Thermal Cycler 480. Optimal conditions for
 cDNA synthesis and thermal cycling can be readily determined by those
 skilled in the art. Conditions which may be found useful include cDNA
 synthesis (60.degree. C., 30 min), 30-45 amplification cycles (94.degree.
 C., 40 sec; 55-70.degree. C., 60 sec), oligo-hybridization (97.degree. C.,
 5 min; 15.degree. C., 5 min; 15.degree. C. soak). The correct reaction
 product contains at least one of the strands of the PCR product and an
 internally hybridized probe.
 C. OH-PCR Product Analysis.
 Amplified reaction products are detected on an LCx.RTM. Analyzer system
 (available from Abbott Laboratories, Abbott Park, Ill.). Briefly, the
 correct reaction product is captured by an antibody labeled microparticle
 at a capturable site on either the PCR product strand or the hybridization
 probe, and the complex is detected by binding of a detectable antibody
 conjugate to either a detectable site on the probe or the PCR strand. Only
 a complex containing a PCR strand hybridized with the internal probe is
 detectable. Detection of a product in urinary tract tissue, in urine, or
 in cells exfoliated into urine comprising a sequence selected from the
 group consisting of SEQUENCE ID NOS 1-13, and fragments or complements
 thereof suggests a diagnosis of urinary tract cancer.
 Many other detection formats exist which can be used and/or modified by
 those skilled in the art to detect the presence of amplified or
 non-amplified nucleic acid sequences including, but not limited to, ligase
 chain reaction (LCR, Abbott Laboratories, Abbott Park, Ill.); Q-beta
 replicase (Gene-Trak.TM., Naperville, Ill.), branched chain reaction
 (Chiron, Emeryville, Calif.) and strand displacement assays (Becton
 Dickinson, Research Triangle Park, N.C.).
 Example 11
 Synthetic Peptide Production
 Synthetic peptides are modeled and then prepared based upon the predicted
 amino acid sequences encoded by the mRNAs of SEQUENCE ID NOS 1-13 (see
 Example 1). In particular, a number of peptides are prepared. All peptides
 are synthesized on a Symphony Peptide Synthesizer (available from Rainin
 Instrument Co, Emeryville, Calif.) or similar instrument, using FMOC
 chemistry, standard cycles and in-situ HBTU activation. Cleavage and
 deprotection conditions are as follows: a volume of 2.5 ml of cleavage
 reagent (77.5% v/v trifluoroacetic acid, 15% v/v ethanedithiol, 2.5% v/v
 water, 5% v/v thioanisole, 1-2% w/v phenol) is added to the resin, and
 agitated at room temperature for 2-4 hours. The filtrate is then removed
 and the peptide is precipitated from the cleavage reagent with cold
 diethyl ether. Each peptide is filtered, purified via reverse-phase
 preparative HPLC using a water/acetonitrile/0.1% TFA gradient, and
 lyophilized. The product is confirmed by mass spectrometry (see Example
 12).
 Disulfide bond formation is accomplished using auto-oxidation conditions,
 as follows: the peptide is dissolved in a minimum amount of DMSO
 (approximately 10 ml) before adding buffer (0.1 M Tris-HCl, pH 6.2) to a
 concentration of 0.3-0.8 mg/ml. The reaction is monitored by HPLC until
 complete formation of the disulfide bond, followed by reverse-phase
 preparative HPLC using a water/acetonitrile/0.1% TFA gradient and
 lyophilization. The product then is confirmed by mass spectrometry (see
 Example 12).
 The purified peptides can be conjugated to Keyhole Limpet Hemocyanin or
 other immunoreactive molecule with glutaraldehyde, mixed with adjuvant,
 and injected into animals.
 Example 12a
 Expression of Protein in a Cell Line Using Plasmid 577
 A. Construction of Expression Plasmids.
 Plasmid 577, described in U.S. patent application Ser. No. 08/478,073,
 filed Jun. 7, 1995 and incorporated herein by reference, has been
 constructed for the expression of secreted antigens in a permanent cell
 line. This plasmid contains the following DNA segments: (a) a 2.3 kb
 fragment of pBR322 containing bacterial beta-lactamase and origin of DNA
 replication; (b) a 1.8 kb cassette directing expression of a neomycin
 resistance gene under control of HSV-1 thymidine kinase promoter and
 poly-A addition signals; (c) a 1.9 kb cassette directing expression of a
 dihydrofolate reductase gene under the control of an Simian Virus 40
 (SV40) promoter and poly-A addition signals; (d) a 3.5 kb cassette
 directing expression of a rabbit immunoglobulin heavy chain signal
 sequence fused to a modified hepatitis C virus (HCV) E2 protein under the
 control of the Simian Virus 40 T-Ag promoter and transcription enhancer,
 the hepatitis B virus surface antigen (HBsAg) enhancer I followed by a
 fragment of Herpes Simplex Virus-1 (HSV-1) genome providing poly-A
 addition signals; and (e) a residual 0.7 kb fragment of SV40 genome late
 region of no function in this plasmid. All of the segments of the vector
 were assembled by standard methods known to those skilled in the art of
 molecular biology.
 Plasmids for the expression of secretable proteins are constructed by
 replacing the hepatitis C virus E2 protein coding sequence in plasmid 577
 with that of a polynucleotide sequence as follows. Digestion of plasmid
 577 with XbaI releases the hepatitis C virus E2 gene fragment. The
 resulting plasmid backbone allows insertion of the cDNA insert downstream
 of the rabbit immunoglobulin heavy chain signal sequence which directs the
 expressed proteins into the secretory pathway of the cell. The cDNA
 fragment is generated by PCR using standard procedures. Encoded in the
 sense PCR primer sequence is an XbaI site, immediately followed by a 12
 nucleotide sequence that encodes the amino acid sequence Ser-Asn-Glu-Leu
 ("SNEL") to promote signal protease processing, efficient secretion and
 final product stability in culture fluids. Immediately following this 12
 nucleotide sequence the primer contains nucleotides complementary to
 template sequences encoding amino acids of the gene. The antisense primer
 incorporates a sequence encoding the following eight amino acids just
 before the stop codons: Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQUENCE ID NO
 16). Within this sequence is incorporated a recognition site to aid in
 analysis and purification of the protein product. A recognition site
 (termed "FLAG") that is recognized by a commercially available monoclonal
 antibody designated anti-FLAG M2 (Eastman Kodak, Co., New Haven, Comm.)
 can be utilized, as well as other comparable sequences and their
 corresponding antibodies. For example, PCR is performed using GeneAmpe
 reagents obtained from Perkin-Elmer-Cetus, as directed by the supplier's
 instructions. PCR primers are used at a final concentration of 0.5 .mu.M.
 PCR is performed on the plasmid template in a 100 .mu.l reaction for 35
 cycles (94.degree. C., 30 seconds; 55.degree. C., 30 seconds; 72.degree.
 C., 90 seconds) followed by an extension cycle of 72.degree. C. for 10
 min.
 B. Transfection of Dihydrofolate Reductase Deficient Chinese Hamster Ovary
 Cells.
 The plasmid described supra is transfected into CHO/dhfr-cells [DXB-111,
 Uriacio et al., Proc Natl Acad Sci USA 77:4451-4466 (1980)]. These cells
 are available from the A.T.C.C., 12301 Parklawn Drive, Rockville, Md.
 20852, under Accession No. CRL 9096. Transfection is performed using the
 cationic liposome-mediated procedure described by P. L. Felgner et al.,
 Proc Natl Acad Sci USA 84:7413-7417 (1987). Particularly, CHO/dhfr-cells
 are cultured in Ham's F-12 media supplemented with 10% fetal calf serum,
 L-glutamine (1 mM) and freshly seeded into a flask at a density of
 5-8.times.10.sup.5 cells per flask. The cells are grown to a confluency of
 between 60 and 80% for transfection. Twenty micrograms (20 .mu.g) of
 plasmid DNA are added to 1.5 ml of Opti-MEM I medium and 100 .mu.l of
 Lipofectin Reagent (Gibco-BRL; Grand Island, N.Y.) are added to a second
 1.5 ml portion of Opti-MEM I media. The two solutions are mixed and
 incubated at room temperature for 20 min. After the culture medium is
 removed from the cells, the cells are rinsed 3 times with 5 ml of Opti-MEM
 I medium. The Opti-MEM I-Lipofection-plasmid DNA solution then is overlaid
 onto the cells. The cells are incubated for 3 hr at 37.degree. C., after
 which time the Opti-MEM I-Lipofectin-DNA solution is replaced with culture
 medium for an additional 24 hr prior to selection.
 C. Selection and Amplification.
 One day after transfection, cells are passaged 1:3 and incubated with
 dhfr/G418 selection medium (hereafter, "F-12 minus medium G"). Selection
 medium is Ham's F-12 with L-glutamine and without hypoxanthine, thymidine
 and glycine (JRH Biosciences, Lenexa, Kans.) and 300 .mu.g per ml G418
 (Gibco-BRL; Grand Island, N.Y.). Media volume-to-surface area ratios of 5
 ml per 25 cm.sup.2 are maintained. After approximately two weeks,
 DHFR/G418 cells are expanded to allow passage and continuous maintenance
 in F-12 minus medium G.
 Amplification of each of the transfected cDNA sequences is achieved by
 stepwise selection of DHFR.sup.+, G418.sup.+ cells with methotrexate
 (reviewed by R. Schimke, Cell 37:705-713 [1984]). Cells are incubated with
 F-12 minus medium G containing 150 nM methotrexate (MTX) (Sigma, St.
 Louis, Mo.) for approximately two weeks until resistant colonies appear.
 Further gene amplification is achieved by selection of 150 nM adapted
 cells with 5 FM MTX.
 D. Antigen Production.
 F-12 minus medium G supplemented with 5 .mu.M MTX is overlaid onto just
 confluent monolayers for 12 to 24 hr at 37.degree. C. in 5% CO.sub.2. The
 growth medium is removed and the cells are rinsed 3 times with Dulbecco's
 phosphate buffered saline (PBS) (with calcium and magnesium) (Gibco-BRL;
 Grand Island, N.Y.) to remove the remaining media/serum which may be
 present. Cells then are incubated with VAS custom medium (VAS custom
 formulation with L-glutamine with HEPES without phenol red, available from
 JRH Bioscience; Lenexa, Kans., product number 52-08678P), for 1 hr at
 37.degree. C. in 5% CO.sub.2. Cells then are overlaid with VAS for
 production at 5 ml per T flask. Medium is removed after seven days of
 incubation, retained, and then frozen to await purification with harvests
 2, 3 and 4. The monolayers are overlaid with VAS for 3 more seven day
 harvests.
 E. Analysis of Antigen Expression.
 Aliquots of VAS supernatants from the cells expressing the protein
 constructs are analyzed, either by SDS-polyacrylamide gel electrophoresis
 (SDS-PAGE) using standard methods and reagents known in the art (Laemmli
 discontinuous gels), or by mass spectrometry.
 F. Purification.
 Purification of the protein containing the FLAG sequence is performed by
 immunoaffinity chromatography using an affinity matrix comprising
 anti-FLAG M2 monoclonal antibody covalently attached to agarose by
 hydrazide linkage (Eastman Kodak Co., New Haven, Conn.). Prior to affinity
 purification, protein in pooled VAS medium harvests from roller bottles is
 exchanged into 50 mM Tris-HCl (pH 7.5), 150 mM NaCl buffer using a
 Sephadex G-25 (Pharmacia Biotech Inc., Uppsala, Sweden) column. Protein in
 this buffer is applied to the anti-FLAG M2 antibody affinity column.
 Non-binding protein is eluted by washing the column with 50 mM Tris-HCl
 (pH 7.5), 150 mM NaCl buffer. Bound protein is eluted using an excess of
 FLAG peptide in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl. The excess FLAG
 peptide can be removed from the purified protein by gel electrophoresis or
 HPLC.
 Although plasmid 577 is utilized in this example, it is known to those
 skilled in the art that other comparable expression systems, such as CMV,
 can be utilized herein with appropriate modifications in reagent and/or
 techniques and are within the skill of the ordinary artisan.
 The largest cloned insert containing the coding region of the gene is then
 sub-cloned into either (i) a eukaryotic expression vector which may
 contain, for example, a cytomegalovirus (CMV) promoter and/or protein
 fusible sequences which aid in protein expression and detection, or (ii) a
 bacterial expression vector containing a superoxide-dismutase (SOD) and
 CMP-KDO synthetase (CKS) or other protein fusion gene for expression of
 the protein sequence. Methods and vectors which are useful for the
 production of polypeptides which contain fusion sequences of SOD are
 described in EPO 0196056, published October 1, 1986, which is incorporated
 herein by reference and those containing fusion sequences of CKS are
 described in EPO Publication No. 0331961, published Sep. 13, 1989, which
 publication is also incorporated herein by reference. This SOD-purified
 protein can be used in a variety of techniques, including, but not limited
 to animal immunization studies, solid phase immunoassays, etc.
 Example 12b
 Expression of Protein in a Cell Line Using pcDNA3.1/Myc-His
 A. Construction of Expression Plasmids.
 Plasmid pcDNA3. 1/Myc-His (Cat.# V855-20, Invitrogen, Carlsbad, Calif.) has
 been constructed, in the past, for the expression of secreted antigens by
 most mammalian cell lines. Expressed protein inserts are fused to a
 myc-his peptide tag. The myc-his tag (SEQUENCE ID NO 17) comprises a c-myc
 oncoprotein epitope and a polyhistidine sequence which are useful for the
 purification of an expressed fusion protein by using either anti-myc or
 anti-his affinity columns, or metalloprotein binding columns.
 Plasmids for the expression of secretable proteins are constructed by
 inserting a polynucleotide sequence selected from the group consisting of
 SEQUENCE ID NOS 1-13, and fragments or complements thereof. Prior to
 construction of an expression plasmid, the cDNA sequence is first cloned
 into a pCR.RTM.-Blunt vector as follows:
 The cDNA fragment is generated by PCR using standard procedures. For
 example, PCR is performed procedures and reagents from Stratageneo, Inc.
 (La Jolla, Calif.), as directed by the manufacturer. PCR primers are used
 at a final concentration of 0.5 .mu.M. PCR using 5 U of pfu polymerase
 (Stratagene, La Jolla, CA) is performed on the plasmid template (see
 Example 2) in a 50 .mu.l reaction for 30 cycles (94.degree. C., 1 min;
 65.degree. C., 1.5 min; 72.degree. C., 3 min) followed by an extension
 cycle of 72.degree. C. for 8 min. (The sense PCR primer sequence comprises
 nucleotides which are either complementary to the pINCY vector directly
 upstream of the gene insert or which incorporate a 5' EcoRI restriction
 site, an adjacent downstream protein translation consensus initiator, and
 a 3' nucleic acid sequence which is the same sense as the 5'-most end of
 the cDNA insert. The antisense PCR primer incorporates a 5' NotI
 restriction sequence and a sequence complementary to the 3' end of the
 cDNA insert just upstream of the 3'-most, in-frame stop codon.) Five
 microliters (5 .mu.l) of the resulting blunted-ended PCR product are
 ligated into 25 ng of linearized pCR.RTM.-Blunt vector (Invitrogen,
 Carlsbad, Calif.) interrupting the lethal ccdB gene of the vector. The
 resulting ligated vector is transformed into TOP10 E. coli (Invitrogen,
 Carlsbad, Calif.) using a One Shot.TM. Transformation Kit (Invitrogen,
 Carlsbad, Calif.) following manufacturer's instructions. The transformed
 cells are grown on LB-Kan (50 .mu.g/ml kanamycin) selection plates at
 37.degree. C. Only cells containing a plasmid with an interrupted ccdB
 gene will grow after transformation [Grant, S. G. N., Proc Natl Acad Sci
 USA 87:4645-4649 (1990)]. Transformed colonies are picked and grown up in
 3 ml of LB-Kan broth at 37.degree. C. Plasmid DNA is isolated by using a
 QIAprep.RTM. (Qiagen Inc., Santa Clarita, Calif.) procedure, as directed
 by the manufacturer. The DNA is cut with EcoRI or SnaBI, and NotI
 restriction enzymes to release the insert fragment. The fragment is run on
 1% Seakem.RTM. LE agarose/0.5 .mu.g/ml ethidium bromide/FE gel, visualized
 by UV irradiation, excised and purified using QIAquick.TM. (Qiagen Inc.,
 Santa Clarita, Calif.) procedures, as directed by the supplier's
 instructions.
 The pcDNA3.1/Myc-His plasmid DNA is linearized by digestion with EcoRI or
 SnaBI, and NotI in the polylinker region of the plasmid DNA. The resulting
 plasmid DNA backbone allows insertion of the purified cDNA fragment,
 supra, downstream of a CMV promoter which directs expression of the
 proteins in mammalian cells. The ligated plasmid is transformed into DH5
 alpham cells (GibcoBRL Grand Island, N.Y.), as directed by the
 manufacturer. Briefly, 10 ng of pcDNA3. 1/Myc-His containing an insert are
 added to 50 .mu.l of competent DH5 alpha cells, and the contents are mixed
 gently. The mixture is incubated on ice for 30 min, heat shocked for 20
 sec at 37.degree. C., and placed on ice for an additional 2 min. Upon
 addition of 0.95 ml of LB medium, the mixture is incubated for 1 hr at
 37.degree. C. while shaking at 225 rpm. The transformed cells then are
 plated onto 100 mm LB/Amp (50 .mu.g/ml ampicillin) plates and grown at
 37.degree. C. Colonies are picked and grown in 3 ml of LB/Amp broth.
 Plasmid DNA is purified using a QlAprep Kit. The presence of the insert is
 confirmed using techniques known to those skilled in the art, including,
 but not limited to restriction digestion and gel analysis. (J. Sambrook et
 al., supra.)
 B. Transfection of Human Embryonic Kidney Cell 293 Cells.
 The expression plasmid described in section A, supra, is retransformed into
 DH5 alpha cells, plated onto LB/ampicillin agar, and grown up in 10 ml of
 LB/ampicillin broth, as described hereinabove. The plasmid is purified
 using a QlAfilteri Maxi Kit (Qiagen, Chatsworth, Calif.) and is
 transfected into HEK293 cells [F. L. Graham et al., J. Gen. Vir. 36:59-72
 (1977)]. These cells are available from the A.T.C.C., 12301 Parklawn
 Drive, Rockville, Md. 20852, under Accession No. CRL 1573. Transfection is
 performed using the cationic lipofectamine-mediated procedure described by
 P. Hawley-Nelson et al., Focus 15.73 (1993). Particularly, HEK293 cells
 are cultured in 10 ml DMEM media supplemented with 10% fetal bovine serum
 (FBS), L-glutamine (2 mM) and freshly seeded into 100 mm culture plates at
 a density of 9.times.10.sup.6 cells per plate. The cells are grown at
 37.degree. C. to a confluency of between 70% and 80% for transfection.
 Eight micrograms (8 .mu.g) of plasmid DNA are added to 800 .mu.l of
 Opti-MEM U.RTM. medium (Gibco-BRL, Grand Island, N.Y.), and 48-96 .mu.l of
 LipofectamineTm Reagent (Gibco-BRL, Grand Island, N.Y.) are added to a
 second 800 .mu.l portion of Opti-MEM I media. The two solutions are mixed
 and incubated at room temperature for 15-30 min. After the culture medium
 is removed from the cells, the cells are washed once with 10 ml of
 serum-free DMEM. The Opti-MEM I-Lipofectamine-plasmid DNA solution is
 diluted with 6.4 ml of serum-free DMEM and then overlaid onto the cells.
 The cells are incubated for 5 hr at 37.degree. C., after which time, an
 additional 8 ml of DMEM with 20% FBS are added. After 18-24 hr, the old
 medium is aspirated, and the cells are overlaid with 5 ml of fresh DMEM
 with 5% FBS. Supernatants and cell extracts are analyzed for gene activity
 72 hr after transfection.
 C. Analysis of Antigen Expression.
 The culture supernatant, supra, is transferred to cryotubes and stored on
 ice. HEK293 cells are harvested by washing twice with 10 ml of cold
 Dulbecco's PBS and lysing by addition of 1.5 ml of CAT lysis buffer
 (Boehringer Mannheim, Indianapolis, Ind.), followed by incubation for 30
 min at room temperature. Lysate is transferred to 1.7 ml polypropylene
 microfuge tubes and centrifuged at 1000.times.g for 10 min. The
 supernatant is transferred to new cryotubes and stored on ice. Aliquots of
 supernatants from the cells and the lysate of the cells expressing the
 protein construct are analyzed for the presence of recombinant protein.
 The aliquots can be run on SDS-polyacrylamide gel electrophoresis
 (SDS-PAGE) using standard methods and reagents known in the art. (J.
 Sambrook et al., supra) These gels can then be blotted onto a solid medium
 such as nitrocellulose, nytran, etc., and the protein band can be
 visualized using Western blotting techniques with anti-myc epitope or
 anti-histidine monoclonal antibodies (Invitrogen, Carlsbad, Calif.) or
 polyclonal serum (see Example 15). Alternatively, the expressed
 recombinant protein can be analyzed by mass spectrometry (see Example 13).
 D. Purification.
 Purification of the recombinant protein containing the myc-his sequence is
 performed using the Xpress.RTM. affinity chromatography system
 (Invitrogen, Carlsbad, Calif.) containing a nickel-charged agarose resin
 which specifically binds polyhistidine residues. Supernatants from
 10.times.100 mm plates, prepared as described supra, are pooled and passed
 over the nickel-charged column. Non-binding protein is eluted by washing
 the column with 50 mM Tris-HCl (pH 7.5)/150 mM NaCl buffer, leaving only
 the myc-his fusion proteins. Bound recombinant protein then is eluted from
 the column using either an excess of imidazole or histidine, or a low pH
 buffer. Alternatively, the recombinant protein can also be purified by
 binding at the myc-his sequence to an affinity column consisting of either
 anti-myc or anti-histidine monoclonal antibodies conjugated through a
 hydrazide or other linkage to an agarose resin and eluting with an excess
 of myc peptide or histidine, respectively.
 The purified recombinant protein can then be covalently cross-linked to a
 solid phase, such as N-hydroxysuccinimide-activated sepharose columns
 (Pharmacia Biotech, Piscataway, N.J.), as directed by supplier's
 instructions. These columns containing covalently linked recombinant
 protein, can then be used to purify anti-antibodies from rabbit or mouse
 sera.
 E. Coating Microtiter Plates with Expressed Proteins.
 Supernatant from a 100 mm plate, as described supra, is diluted in an
 appropriate volume of PBS. Then, 100 .mu.l of the resulting mixture is
 placed into each well of a Reacti-Bind.TM. metal chelate microtiter plate
 (Pierce, Rockford, Ill.), incubated at room temperature while shaking, and
 followed by three washes with 200 .mu.l each of PBS with 0.05% Tween.RTM.
 20. The prepared microtiter plate can then be used to screen polyclonal
 antisera for the presence of antibodies (see Example 17).
 Although pcDNA3.1/Myc-His is utilized in this example, it is known to those
 skilled in the art that other comparable expression systems can be
 utilized herein with appropriate modifications in reagent and/or
 techniques and are within the skill of one of ordinary skill in the art.
 The largest cloned insert containing the coding region of the gene is
 sub-cloned into either (i) a eukaryotic expression vector which may
 contain, for example, a cytomegalovirus (CMV) promoter and/or protein
 fusible sequences which aid in protein expression and detection, or (ii) a
 bacterial expression vector containing a superoxide-dismutase (SOD) and
 CMP-KDO synthetase (CKS) or other protein fusion gene for expression of
 the protein sequence. Methods and vectors which are useful for the
 production of polypeptides which contain fusion sequences of SOD are
 described in published EPO application No. EP 0 196 056, published Oct. 1,
 1986, which is incorporated herein by reference, and vectors containing
 fusion sequences of CKS are described in published EPO application No. EP
 0 331 961, published Sep. 13, 1989, which publication is also incorporated
 herein by reference. The purified protein can be used in a variety of
 techniques, including, but not limited to animal immunization studies,
 solid phase immunoassays, etc.
 Example 13
 Chemical Analysis of Proteins
 A. Analysis of Tryptic Peptide Fragments Using MS.
 Sera from patients with urinary tract disease, such as urinary tract
 cancer, sera from patients with no urinary tract disease, extracts of
 urinary tract tissues or cells from patients with urinary tract disease,
 such as urinary tract cancer, extracts of urinary tract tissues or cells
 from patients with no urinary tract disease, exfoliated urinary tract
 cells in urine, and extracts of tissues or cells from other non-diseased
 or diseased organs of patients are run on a polyacrylamide gel using
 standard procedures and stained with Coomassie Blue. Sections of the gel
 suspected of containing a target polypeptide are excised and subjected to
 an in-gel reduction, acetamidation and tryptic digestion. P. Jeno et al.,
 Anal. Bio. 224:451-455 (1995) and J. Rosenfeld et al., Anal. Bio.
 203:173-179 (1992). The gel sections are washed with 100 mM NH.sub.4
 HCO.sub.3 and acetonitrile. The shrunken gel pieces are swollen in
 digestion buffer (50 mM NH.sub.4 HCO.sub.3, 5 mM CaCl.sub.2 and 12.5
 .mu.g/ml trypsin) at 4.degree. C. for 45 min. The supernatant is aspirated
 and replaced with 5 to 10 .mu.l of digestion buffer without trypsin and
 allowed to incubate overnight at 37.degree. C. Peptides are extracted with
 3 changes of 5% formic acid and acetonitrile and evaporated to dryness.
 The peptides are adsorbed to approximately 0.1 .mu.l of POROS R2 sorbent
 (Perseptive Biosystems, Framingham, Mass.) trapped in the tip of a drawn
 gas chromatography capillary tube by dissolving them in 10 .mu.l of 5%
 formic acid and passing it through the capillary. The adsorbed peptides
 are washed with water and eluted with 5% formic acid in 60% methanol. The
 eluant is passed directly into the spraying capillary of an API III mass
 spectrometer (Perkin-Elmer Sciex, Thornhill, Ontario, Canada) for analysis
 by nano-electrospray mass spectrometry. M. Wilm et al., Int. J. Mass
 Spectrom. Ion Process 136:167-180 (1994) and M. Wilm et al., Anal. Chem.
 66:1-8 (1994). The masses of the tryptic peptides are determined from the
 mass spectrum obtained off the first quadrupole. Masses corresponding to
 predicted peptides can be further analyzed in MS/MS mode to give the amino
 acid sequence of the peptide.
 B. Peptide Fragment Analysis Using LC/MS.
 The presence of polypeptides predicted from mRNA sequences found in
 hyperplastic disease tissues also can be confirmed using liquid
 chromatography/tandem mass spectrometry (LC/MS/MS). D. Hess et al.,
 METHODS. A Companion to Methods in Enzymology 6:227-238 (1994). The serum
 specimen or tumor extract from the patient is denatured with SDS and
 reduced with dithiothreitol (1.5 mg/ml) for 30 min at 90.degree. C.
 followed by alkylation with iodoacetamide (4 mg/ml) for 15 min at
 25.degree. C. Following acrylamide electrophoresis, the polypeptides are
 electroblotted to a cationic membrane and stained with Coomassie Blue.
 Following staining, the membranes are washed and sections thought to
 contain the unknown polypeptides are cut out and dissected into small
 pieces. The membranes are placed in 500 .mu.l microcentrifuge tubes and
 immersed in 10 to 20 .mu.l of proteolytic digestion buffer (100 mM
 Tris-HCl, pH 8.2, containing 0.1 M NaCl, 10% acetonitrile, 2 mM CaCI.sub.2
 and 5 .mu.g/ml trypsin) (Sigma, St. Louis, Mo.). After 15 hr at 37.degree.
 C., 3 .mu.l of saturated urea and 1 .mu.l of 100 .mu.g/ml trypsin are
 added and incubated for an additional 5 hr at 37.degree. C. The digestion
 mixture is acidified with 3 .mu.l of 10% trifluoroacetic acid and
 centrifuged to separate supernatant from membrane. The supernatant is
 injected directly onto a microbore, reverse phase HPLC column and eluted
 with a linear gradient of acetonitrile in 0.05% trifluoroacetic acid. The
 eluate is fed directly into an electrospray mass spectrometer, after
 passing though a stream splitter if necessary to adjust the volume of
 material. The data is analyzed following the procedures set forth in
 Example 13, Section A.
 Example 14
 Gene Immunization Protocol
 A. In vivo Antigen Expression.
 Gene immunization circumvents protein purification steps by directly
 expressing an antigen in vivo after inoculation of the appropriate
 expression vector. Also, production of antigen by this method may allow
 correct protein folding and glycosylation since the protein is produced in
 mammalian tissue. The method utilizes insertion of the gene sequence into
 a plasmid which contains a CMV promoter, expansion and purification of the
 plasmid and injection of the plasmid DNA into the muscle tissue of an
 animal. Preferred animals include mice and rabbits. See, for example, H.
 Davis et al., Human Molecular Genetics 2:1847-1851 (1993). After one or
 two booster immunizations, the animal can then be bled, ascites fluid
 collected, or the animal's spleen can be harvested for production of
 hybridomas.
 B. Plasmid Preparation and Purification.
 cDNA sequences are generated from the cDNA-containing vector using
 appropriate PCR primers containing suitable 5' restriction sites. The PCR
 product is cut with appropriate restriction enzymes and inserted into a
 vector which contains the CMV promoter (for example, pRc/CMV or pcDNA3
 vectors from Invitrogen, San Diego, Calif.). This plasmid then is expanded
 in the appropriate bacterial strain and purified from the cell lysate
 using a CsCl gradient or a Qiagen plasmid DNA purification column. All
 these techniques are familiar to one of ordinary skill in the art of
 molecular biology.
 C. Immunization Protocol.
 Anesthetized animals are immunized intramuscularly with 0.1-100 jg of the
 purified plasmid diluted in PBS or other DNA uptake enhancers
 (Cardiotoxin, 25% sucrose). See, for example, H. Davis et al., Human Gene
 Therapy 4:733-740 (1993); and P. W. Wolff et al., Biotechniques 11:474-485
 (1991). One to two booster injections are given at monthly intervals.
 D. Testing and Use of Antiserum.
 Animals are bled and the resultant sera tested for antibody using peptides
 synthesized from the known gene sequence (see Example 16) using techniques
 known in the art, such as Western blotting or EIA techniques. Antisera
 produced by this method can then be used to detect the presence of the
 antigen in a patient's tissue or cell extract or in a patient's serum by
 ELISA or Western blotting techniques, such as those described in Examples
 15 through 18.
 Example 15
 Production of Antibodies
 A. Production of Polyclonal Antisera.
 Antiserum against polypeptides is prepared by injecting appropriate animals
 with peptides whose sequences are derived from that of the predicted amino
 acid sequence of the nucleotide sequence. The synthesis of peptides is
 described in Example 11. Peptides used as immunogen either can be
 conjugated to a carrier such as keyhole limpet hemocyanine (KLH), prepared
 as described hereinbelow, or unconjugated (i.e., not conjugated to a
 carrier such as KLH).
 1. Peptide Conjugation.
 Peptide is conjugated to maleimide activated keyhole limpet hemocyanine
 (KLH, commercially available as Imjecto, available from Pierce Chemical
 Company, Rockford, Ill.). Imject.RTM. contains about 250 moles of reactive
 maleimide groups per mole of hemocyanine. The activated KLH is dissolved
 in phosphate buffered saline (PBS, pH 8.4) at a concentration of about 7.7
 mg/ml. The peptide is conjugated through cysteines occurring in the
 peptide sequence, or to a cysteine previously added to the synthesized
 peptide in order to provide a point of attachment. The peptide is
 dissolved in dimethyl sulfoxide (DMSO, Sigma Chemical Company, St. Louis,
 Mo.) and reacted with the activated KLH at a mole ratio of about 1.5 moles
 of peptide per mole of reactive maleimide attached to the KLH. A procedure
 for the conjugation of peptide is provided hereinbelow. It is known to the
 ordinary artisan that the amounts, times and conditions of such a
 procedure can be varied to optimize peptide conjugation.
 The conjugation reaction described hereinbelow is based on obtaining 3 mg
 of KLH peptide conjugate ("conjugated peptide"), which contains about 0.77
 moles of reactive maleimide groups. This quantity of peptide conjugate
 usually is adequate for one primary injection and four booster injections
 for production of polyclonal antisera in a rabbit. Briefly, peptide is
 dissolved in DMSO at a concentration of 1.16 .mu.moles/100 .mu.l of DMSO.
 One hundred microliters (100 .mu.l) of the DMSO solution are added to 380
 .mu.l of the activated KLH solution prepared as described hereinabove, and
 20 .mu.l of PBS (pH 8.4) are added to bring the volume to 500 .mu.l. The
 reaction is incubated overnight at room temperature with stirring. The
 extent of reaction is determined by measuring the amount of unreacted
 thiol in the reaction mixture. The difference between the starting
 concentration of thiol and the final concentration is assumed to be the
 concentration of peptide which has coupled to the activated KLH. The
 amount of remaining thiol is measured using Ellman's reagent
 (5,5'-dithiobis(2-nitrobenzoic acid), Pierce Chemical Company, Rockford,
 Ill.). Cysteine standards are made at a concentration of 0, 0.1, 0.5, 2, 5
 and 20 mM by dissolving 35 mg of cysteine HCl (Pierce Chemical Company,
 Rockford, Ill.) in 10 ml of PBS (pH 7.2) and diluting the stock solution
 to the desired concentration(s). The photometric determination of the
 concentration of thiol is accomplished by placing 200 .mu.l of PBS (pH
 8.4) in each well of an Immulon 2.RTM. microwell plate (Dynex
 Technologies, Chantilly, Va.). Next, 10 .mu.l of standard or reaction
 mixture is added to each well. Finally, 20 .mu.l of Ellman's reagent at a
 concentration of 1 mg/ml in PBS (pH 8.4) is added to each well. The wells
 are incubated for 10 minutes at room temperature, and the absorbance of
 all wells is read at 415 nm with a microplate reader (such as the BioRad
 Model 3550, BioRad, Richmond, Calif.). The absorbance of the standards is
 used to construct a standard curve and the thiol concentration of the
 reaction mixture is determined from the standard curve. A decrease in the
 concentration of free thiol is indicative of a successful conjugation
 reaction. Unreacted peptide is removed by dialysis against PBS (pH 7.2) at
 room temperature for 6 hours. The conjugate is stored at 2-8.degree. C. if
 it is to be used immediately; otherwise, it is stored at -20.degree. C. or
 colder.
 2. Animal Immunization.
 Female white New Zealand rabbits weighing 2 kg or more are used for raising
 polyclonal antiserum. Generally, one animal is immunized per unconjugated
 or conjugated peptide (prepared as described hereinabove). One week prior
 to the first immunization, 5 to 10 ml of blood is obtained from the animal
 to serve as a non-immune prebleed sample.
 Unconjugated or conjugated peptide is used to prepare the primary immunogen
 by emulsifying 0.5 ml of the peptide at a concentration of 2 mg/ml in PBS
 (pH 7.2) which contains 0.5 ml of complete Freund's adjuvant (CFA) (Difco,
 Detroit, Mich.). The immunogen is injected into several sites of the
 animal via subcutaneous, intraperitoneal, and/or intramuscular routes of
 administration. Four weeks following the primary immunization, a booster
 immunization is administered. The immunogen used for the booster
 immunization dose is prepared by emulsifying 0.5 ml of the same
 unconjugated or conjugated peptide used for the primary immunogen, except
 that the peptide now is diluted to 1 mg/ml with 0.5 ml of incomplete
 Freund's adjuvant (IFA) (Difco, Detroit, Mich.). Again, the booster dose
 is administered into several sites and can utilize subcutaneous,
 intraperitoneal and intramuscular types of injections. The animal is bled
 (5 ml) two weeks after the booster immunization and the serum is tested
 for immunoreactivity to the peptide, as described below. The booster and
 bleed schedule is repeated at 4 week intervals until an adequate titer is
 obtained. The titer or concentration of antiserum is determined by
 microtiter EIA as described in Example 18, below. An antibody titer of
 1:500 or greater is considered an adequate titer for further use and
 study.
 B. Production of Monoclonal Antibody.
 1. Immunization Protocol.
 Mice are immunized using immunogens prepared as described hereinabove,
 except that the amount of the unconjugated or conjugated peptide for
 monoclonal antibody production in mice is one-tenth the amount used to
 produce polyclonal antisera in rabbits. Thus, the primary immunogen
 consists of 100 .mu.g of unconjugated or conjugated peptide in 0.1 ml of
 CFA emulsion; while the immunogen used for booster immunizations consists
 of 50 .mu.g of unconjugated or conjugated peptide in 0.1 ml of IFA.
 Hybridomas for the generation of monoclonal antibodies are prepared and
 screened using standard techniques. The methods used for monoclonal
 antibody development follow procedures known in the art such as those
 detailed in Kohler and Milstein, Nature 256:494 (1975) and reviewed in J.
 G. R. Hurrel, ed., Monoclonal Hybridoma Antibodies: Techniques and
 Applications, CRC Press, Inc., Boca Raton, Fla. (1982). Another method of
 monoclonal antibody development which is based on the Kohler and Milstein
 method is that of L. T. Mimms et al., Virology 176:604-619 (1990), which
 is incorporated herein by reference.
 The immunization regimen (per mouse) consists of a primary immunization
 with additional booster immunizations. The primary immunogen used for the
 primary immunization consists of 100 .mu.g of unconjugated or conjugated
 peptide in 50 .mu.l of PBS (pH 7.2) previously emulsified in 50 .mu.l of
 CFA. Booster immunizations performed at approximately two weeks and four
 weeks post primary immunization consist of 50 .mu.g of unconjugated or
 conjugated peptide in 50 .mu.l of PBS (pH 7.2) emulsified with 50 .mu.l
 IFA. A total of 100 .mu.l of this immunogen is inoculated
 intraperitoneally and subcutaneously into each mouse. Individual mice are
 screened for immune response by microtiter plate enzyme immunoassay (EIA)
 as described in Example 18 approximately four weeks after the third
 immunization. Mice are inoculated either intravenously, intrasplenically
 or intraperitoneally with 50 gg of unconjugated or conjugated peptide in
 PBS (pH 7.2) approximately fifteen weeks after the third immunization.
 Three days after this intravenous boost, splenocytes are fused with, for
 example, Sp2/0-Ag14 myeloma cells (Milstein Laboratories, England) using
 the polyethylene glycol (PEG) method. The fusions are cultured in Iscove's
 Modified Dulbecco's Medium (IMDM) containing 10% fetal calf serum (FCS),
 plus 1% hypoxanthine, aminopterin and thymidine (HAT). Bulk cultures are
 screened by microtiter plate EIA following the protocol in Example 18.
 Clones reactive with the peptide used an immunogen and non-reactive with
 other peptides (i.e., peptides of a protein not used as the immunogen) are
 selected for final expansion. Clones thus selected are expanded, aliquoted
 and frozen in IMDM containing 10% FCS and 10% dimethyl-sulfoxide.
 2. Production of Ascites Fluid Containing Monoclonal Antibodies.
 Frozen hybridoma cells prepared as described hereinabove are thawed and
 placed into expansion culture. Viable hybridoma cells are inoculated
 intraperitoneally into Pristane treated mice. Ascitic fluid is removed
 from the mice, pooled, filtered through a 0.2 i filter and subjected to an
 immunoglobulin class G (IgG) analysis to determine the volume of the
 Protein A column required for the purification.
 3. Purification of Monoclonal Antibodies From Ascites Fluid.
 Briefly, filtered and thawed ascites fluid is mixed with an equal volume of
 Protein A sepharose binding buffer (1.5 M glycine, 3.0 M NaCl, pH 8.9) and
 refiltered through a 0.2 g filter. The volume of the Protein A column is
 determined by the quantity of IgG present in the ascites fluid. The eluate
 then is dialyzed against PBS (pH 7.2) overnight at 2-8.degree. C. The
 dialyzed monoclonal antibody is sterile filtered and dispensed in
 aliquots. The immunoreactivity of the purified monoclonal antibody is
 confirmed by determining its ability to specifically bind to the peptide
 used as the immunogen by use of the EIA microtiter plate assay procedure
 of Example 18. The specificity of the purified monoclonal antibody is
 confirmed by determining its lack of binding to irrelevant peptides such
 as peptides not used as the immunogen. The purified anti-monoclonal thus
 prepared and characterized is placed at either 2-8.degree. C. for short
 term storage or at -80.degree. C. for long term storage.
 4. Further Characterization of Monoclonal Antibody.
 The isotype and subtype of the monoclonal antibody produced as described
 hereinabove can be determined using commercially available kits (available
 from Amersham. Inc., Arlington Heights, Ill.). Stability testing also can
 be performed on the monoclonal antibody by placing an aliquot of the
 monoclonal antibody in continuous storage at 2-8.degree. C. and assaying
 optical density (OD) readings throughout the course of a given period of
 time.
 C. Use of Recombinant Proteins as Immunogens.
 It is within the scope of the present invention that recombinant proteins
 made as described herein can be utilized as immunogens in the production
 of polyclonal and monoclonal antibodies, with corresponding changes in
 reagents and techniques known to those skilled in the art.
 Example 16
 Purification of Serum Antibodies Which Specifically Bind to Synthetic
 Peptides
 Immune sera, obtained, for example, as described hereinabove in Examples 14
 and/or 15, is affinity purified using immobilized synthetic peptides
 prepared as described in Example 11, or recombinant proteins prepared as
 described in Example 12. An IgG fraction of the antiserum is obtained by
 passing the diluted, crude antiserum over a Protein A column (Affi-Gel
 protein A, Bio-Rad, Hercules, Calif.). Elution with a buffer (Binding
 Buffer, supplied by the manufacturer) removes substantially all proteins
 that are not immunoglobulins. Elution with 0.1M buffered glycine (pH 3)
 gives an immunoglobulin preparation that is substantially free of albumin
 and other serum proteins.
 Immunoaffinity chromatography is performed to obtain a preparation with a
 higher fraction of specific antigen-binding antibody. The peptide used to
 raise the antiserum is immobilized on a chromatography resin, and the
 specific antibodies directed against its epitopes are adsorbed to the
 resin. After washing away non-binding components, the specific antibodies
 are eluted with 0.1 M glycine buffer, pH 2.3. Antibody fractions are
 immediately neutralized with 1.OM Tris buffer (pH 8.0) to preserve
 immunoreactivity. The chromatography resin chosen depends on the reactive
 groups present in the peptide. If the peptide has an amino group, a resin
 such as Affi-Gel 10 or Affi-Gel 15 is used (Bio-Rad, Hercules, Calif.). If
 coupling through a carboxy group on the peptide is desired, Affi-Gel 102
 can be used (Bio-Rad, Hercules, Calif.). If the peptide has a free
 sulfhydryl group, an organomercurial resin such as Affi-Gel 501 can be
 used (Bio-Rad, Hercules, Calif.).
 Alternatively, spleens can be harvested and used in the production of
 hybridomas to produce monoclonal antibodies following routine methods
 known in the art as described hereinabove.
 Example 17
 Western Blotting of Tissue Samples
 Protein extracts are prepared by homogenizing tissue samples in 0.1 M
 Tris-HCl (pH 7.5), 15% (w/v) glycerol, 0.2mM EDTA, 1.0 mM
 1,4-dithiothreitol, 10 .mu.g/ml leupeptin and 1.0 mM
 phenylmethylsulfonylfluoride [Kain et al., Biotechniques, 17:982 (1994)].
 Following homogenization, the homogenates are centrifuged at 4.degree. C.
 for 5 minutes to separate supernatant from debris. Debris is reextracted
 by homogenization with a buffer that is similar to above also contains 0.1
 M Tricine and 0.1% SDS. The supernatant from the second extraction is used
 for Western blotting. For protein quantitation, 2-5 .mu.l of supernatant
 are added to 1.5 ml of Coomassie Protein Reagent (Pierce, Rockford, Ill.),
 and the resulting absorbance at 595 nm is measured.
 For SDS-PAGE, samples are adjusted to desired protein concentration with
 Tricine Buffer (Novex, San Diego, Calif.), mixed with an equal volume of
 2X Tricine sample buffer (Novex, San Diego, Calif.), and heated for 5
 minutes at 100.degree. C. in a thermal cycler. Samples are then applied to
 a Novex 10-20% Precast Tricine Gel for electrophoresis. Following
 electrophoresis, samples are transferred from the gels to nitrocellulose
 membranes in Novex Tris-Glycine Transfer buffer. Membranes are then probed
 with specific anti-peptide antibodies using the reagents and procedures
 provided in the Western Lights or Western Lights Plus (Tropix, Bedford,
 Mass.) chemiluminesence detection kits. Chemiluminesent bands are
 visualized by exposing the developed membranes to Hyperfilm ECL (Amersham,
 Arlington Heights, Ill.).
 Competition experiments are performed in an analogous manner as above, with
 the following exception; the primary antibodies (anti-peptide polyclonal
 antisera) are pre-incubated for 30 minutes at room temperature with
 varying concentrations of peptide immunogen prior to exposure to the
 nitrocellulose filter. Development of the Western is performed as above.
 After visualization of the bands on film, the bands can also be visualized
 directly on the membranes by the addition and development of a chromogenic
 substrate such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP). This
 chromogenic solution contains 0.016% BCIP in a solution containing 100 mM
 NaCl, 5 mM MgCl2 and 100 mM Tris-HCl (pH 9.5). The filter is incubated in
 the solution at room temperature until the bands develop to the desired
 intensity. Molecular mass determination is made based upon the mobility of
 pre-stained molecular weight standards (Novex, San Diego, Calif.) or
 biotinylated molecular weight standards (Tropix, Bedford, Mass.).
 Example 18
 EIA Microtiter Plate Assay
 The immunoreactivity of antiserum preferably obtained from rabbits or mice
 as described in Examples 14, 15, or 16 is determined by means of a
 microtiter plate EIA, as follows. Briefly, synthetic peptides prepared as
 described in Example 10, are dissolved in 50 mM carbonate buffer (pH 9.6)
 to a final concentration of 2 .mu.g/ml. Next, 100 .mu.l of the peptide or
 protein solution are placed in each well of an Inmulon 2.RTM. microtiter
 plate (Dynex Technologies, Chantilly, Va.). The plate is incubated
 overnight at room temperature and then washed four times with deionized
 water. The wells are blocked by adding 125 .mu.l of a suitable protein
 blocking agent, such as Superblock.RTM. (Pierce Chemical Company,
 Rockford, Ill.), in phosphate buffered saline (PBS, pH 7.4) to each well
 and then immediately discarding the solution. This blocking procedure is
 performed three times. Antiserum obtained from immunized rabbits or mice
 prepared as previously described is diluted in a protein blocking agent
 (e.g., a 3% Superblock.RTM. solution) in PBS containing 0.05%
 Tween-20.RTM. (monolaurate polyoxyethylene ether) (Sigma Chemical Company,
 St. Louis, Mo.) and 0.05% sodium azide at dilutions of 1:100, 1:500,
 1:2500, 1:12,500, and 1:62,500 and placed in each well of the coated
 microtiter plate. The wells then are incubated for three hours at room
 temperature. Each well is washed four times with deionized water. One
 hundred t of alkaline phosphatase-conjugated goat anti-rabbit IgG or goat
 anti-mouse IgG antiserum (Southern Biotech, Birmingham, AB), diluted
 1:2000 in 3% Superblock.RTM. solution in phosphate buffered saline
 containing 0.05% Tween 20.RTM. and 0.05% sodium azide, is added to each
 well . The wells are incubated for two hours at room temperature. Next,
 each well is washed four times with deionized water. One hundred
 microliters (100 RI) of paranitrophenyl phosphate substrate (Kirkegaard
 and Perry Laboratories, Gaithersburg, Md.) then are added to each well.
 The wells are incubated for thirty minutes at room temperature. The
 absorbance at 405 nm is read of each well. Positive reactions are
 identified by an increase in absorbance at 405 nm in the test well above
 that absorbance given by a non-immune serum (negative control). A positive
 reaction is indicative of the presence of detectable antibodies. Titers of
 the anti-peptide antisera are calculated from the previously described
 dilutions of antisera and defined as the calculated dilution, where
 A.sub.405 nm =0.5 OD.
 In addition to titers, apparent affinities [K.sub.d (app)] may also be
 determined for some of the anti-peptide antisera. EIA microtiter plate
 assay results can be used to derive the apparent dissociation constants
 (K.sub.d) based on an analog of the Michaelis-Menten equation [V. Van
 Heyningen, Methods in Enzymology, Vol.121, p. 472 (1986) and further
 described in X. Qiu, et al., Journal of Immunology, Vol. 156, p. 3350
 (1996)]:
 ##EQU1##
 Where [Ag-Ab] is the antigen-antibody complex concentration,
 [Ag-Ab].sub.max is the maximum complex concentration, [Ab] is the antibody
 concentration, and K.sub.d is the dissociation constant. During the curve
 fitting, the [Ag-Ab] is replaced with the background subtracted value of
 the OD.sub.405 nm at the given concentration of Ab. Both K.sub.d and
 [OD.sub.405 nm ].sub.max, which corresponds to the [Ag-Ab].sub.max, are
 treated as fitted parameters. A software program, for example, Origin.TM.,
 can be used for the curve fitting.
 Example 19
 Coating of Solid Phase Particles
 A. Coating of Microparticles with Antibodies Which Specifically Bind to
 Antigen.
 Affinity purified antibodies which specifically bind to protein (see
 Example 15) are coated onto microparticles of polystyrene, carboxylated
 polystyrene, polymethylacrylate or similar particles having a radius in
 the range of about 0.1 to 20 .mu.m. Microparticles may be either passively
 or actively coated. One coating method comprises coating EDAC
 (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (Aldrich
 Chemical Co., Milwaukee, Wis.) activated carboxylated latex microparticles
 with antibodies which specifically bind to protein, as follows. Briefly, a
 final 0.375% solid suspension of resin washed carboxylated latex
 microparticles (available from Bangs Laboratories, Carmel, Ind. or
 Serodyn, Indianapolis, Ind.) are mixed in a solution containing 50 mM MES
 buffer, pH 4.0 and 150 mg/l of affinity purified anti-protein antibody
 (see Example 14) for 15 min in an appropriate container. EDAC coupling
 agent is added to a final concentration of 5.5 .mu.g/ml to the mixture and
 mixed for 2.5 hr at room temperature.
 The microparticles then are washed with 8 volumes of a Tween 20.RTM./sodium
 phosphate wash buffer (pH 7.2) by tangential flow filtration using a 0.2
 .mu.m Microgon Filtration module. Washed microparticles are stored in an
 appropriate buffer which usually contains a dilute surfactant and
 irrelevant protein as a blocking agent, until needed.
 B. Coating of 1/4 Inch Beads.
 Antibodies which specifically bind to the specific antigen also may be
 coated on the surface of 1/4 inch polystyrene beads by routine methods
 known in the art (Snitman et al., U.S. Pat. No. 5,273,882, incorporated
 herein by reference) and used in competitive binding or EIA sandwich
 assays.
 Polystyrene beads first are cleaned by ultrasonicating them for about 15
 seconds in 10 mM NaHCO3 buffer at pH 8.0. The beads then are washed in
 deionized water until all fines are removed. Beads then are immersed in an
 antibody solution in 10 mM carbonate buffer, pH 8 to 9.5. The antibody
 solution can be as dilute as 1 .mu.g/ml in the case of high affinity
 monoclonal antibodies or as concentrated as about 500 .mu.g/ml for
 polyclonal antibodies which have not been affinity purified. Beads are
 coated for at least 12 hours at room temperature, and then they are washed
 with deionized water. Beads may be air dried or stored wet (in PBS, pH
 7.4). They also may be overcoated with protein stabilizers (such as
 sucrose) or protein blocking agents used as non-specific binding blockers
 (such as irrelevant proteins, Carnation skim milk, Superblock.RTM., or the
 like).
 Example 20
 Microparticle Enzyme Immunoassay (MEIA)
 Specific antigens are detected in patient test samples by performing a
 standard antigen competition EIA or antibody sandwich EIA and utilizing a
 solid phase such as microparticles (MEIA). The assay can be performed on
 an automated analyzer such as the IMx.RTM. Analyzer (Abbott Laboratories,
 Abbott Park, Ill.).
 A. Antibody Sandwich EIA.
 Briefly, samples suspected of containing antigen are incubated in the
 presence of specific antibody-coated microparticles (prepared as described
 in Example 19) in order to form antigen/antibody complexes. The
 microparticles then are washed and an indicator reagent comprising an
 antibody conjugated to a signal generating compound (i.e., enzymes such as
 alkaline phosphatase or horseradish peroxide) is added to the
 antigen/antibody complexes or the microparticles and incubated. The
 microparticles are washed and the bound antibody/antigen/antibody
 complexes are detected by adding a substrate (e.g., 4-methyl umbelliferyl
 phosphate (MUP), or OPD/peroxide, respectively), that reacts with the
 signal generating compound to generate a measurable signal. An elevated
 signal in the test sample, compared to the signal generated by a negative
 control, detects the presence of antigen. The presence of antigen in the
 test sample is indicative of a diagnosis of bladder cancer.
 B. Competitive Binding Assay.
 The competitive binding assay uses a peptide or protein that generates a
 measurable signal when the labeled peptide is contacted with an
 anti-peptide antibody coated microparticle. This assay can be performed on
 the IMx.RTM. Analyzer (available from Abbott Laboratories, Abbott Park,
 Ill.). The labeled peptide is added to the specific antibody-coated
 microparticles (prepared as described in Example 17) in the presence of a
 test sample suspected of containing the antigen, and incubated for a time
 and under conditions sufficient to form labeled peptide (or labeled
 protein)/bound antibody complexes and/or patient antigen/bound antibody
 complexes. The antigen in the test sample competes with the labeled
 peptide (or protein) for binding sites on the microparticle, specific
 antigen in the test sample results in a lowered binding of labeled peptide
 and antibody coated microparticles in the assay since antigen in the test
 sample and the peptide or protein compete for antibody binding sites. A
 lowered signal (compared to a control) indicates the presence of specific
 antigen in the test sample. The presence of antigen derived from a
 polypeptide encoded by the polynucleotides comprising SEQUENCE ID NOS
 1-13, and fragments or complements, thereof in bladder tissue suggests the
 diagnosis of bladder cancer.
 The polynucleotides and the proteins encoded thereby which are provided and
 discussed hereinabove are useful as markers of urinary tract cancer. Tests
 based upon the appearance of this marker in a test sample such as
 exfoliated cells in urine or micrometastases in blood can provide low
 cost, non-invasive, diagnostic information to aid the physician to make a
 diagnosis of cancer, to help select a therapy protocol, or to monitor the
 success of a chosen therapy. This marker may appear in readily accessible
 body fluids such as blood or urine as antigens derived from the diseased
 tissue which are detectable by immunological methods.
 Example 21
 Immunohistochemical Detection of Protein
 Antiserum against a synthetic peptide described in Example 15, above, is
 used to immunohistochemically stain a variety of normal and diseased
 tissues using standard proceedures. Briefly, frozen blocks of tissue are
 cut into 6 micron sections, and placed on microscope slides. After
 fixation in cold acetone, the sections are dried at room temperature, then
 washed with phosphate buffered saline and blocked. The slides are
 incubated with the antiserum against a synthetic peptide derived from the
 peptide sequence at a dilution of 1:500, washed, incubated with
 biotinylated goat anti-rabbit antibody, washed again, and incubated with
 avidin labeled with horseradish peroxidase. After a final wash, the slides
 are incubated with 3-amino-9-ethylcarbazole substrate which gives a red
 stain. The slides are counterstained with hematoxylin, mounted, and
 examined under a microscope by a pathologist.