Human insulinoma-associated cDNA

A novel insulinoma-associated, neuroendocrine tumor-associated cDNA sequence is disclosed. The sequence and fragments thereof are useful for the diagnosis and identification of insulinoma and neuroendocrine tumors. The invention relates to a method for identifying a cancer employing the insulinoma-associated nucleic acid, polypeptide and antibody generated thereto.

FIELD OF THE INVENTION
 The present invention relates to the field of cancer diagnosis and therapy.
 In particular, this invention relates to the isolation and use of a novel
 insulinoma-associated neuroendocrine tumor-associated cDNA, polypeptide
 and antibody generated thereto for research and diagnostic purposes.
 BACKGROUND OF THE INVENTION
 The pancreas contains cells of both the exocrine and endocrine system.
 Endocrine cells are found scattered throughout the pancreas in clusters of
 cells termed the islets of Langerhans, or pancreatic islets. Histological
 staining and analysis reveal several cell types within the islets. Two of
 the most prevalent cells are the alpha cells which constitute
 approximately 20 percent of the islet cells, and the beta cells which
 constitute about 75 percent of the islet cells. Other cells types within
 the islet include the delta cell and the pancreatic polypeptide cell.
 While beta cells comprise the major cell type present in the pancreatic
 islets, beta cells are less than 2% of the total pancreatic mass. Beta
 cells are the only cell type in the body that secrete insulin hormone to
 regulate glucose metabolism. These cells and other endocrine cell types of
 the pancreatic islets release their hormones directly into the
 circulation.
 Endocrine cells of the pancreas share similarities with neuroendocrine
 tissue including cells of the pituitary gland, thyroid medulla,
 parathyroid, carotid body, adrenal medulla and small cells of the lung. It
 is generally accepted that pancreatic islet cells express a number of
 neuroectodermal markers. These markers include neuronal-specific enolase
 synaptophysin, and tyrosine hydroxylase (Polak et al., 1984 In Evolution
 and Tumor Pathology of the Neuroendocrine System. S. Falkmer et al., Eds.
 Elsevier, Amsterdam, p. 433; Weidenmann et al., Proc. Natl. Acad. Sci.,
 USA 83:3500-3504 (1986); and Teitelman et al., Devel. Biol. 121:454-466
 (1987) respectively). Dissociated pancreatic islet cells can extend
 neurite-like processes in vitro and these processes contain neurofilament
 protein (Teitelman, Devel. Biol. 142:368-379, 1990).
 The incidence of pancreatic cancer in industrialized countries has
 increased over the past twenty years. The majority of pancreatic cancers
 have morphologic characteristics of cells of the exocrine pancreas,
 however some pancreatic cancers are derived from beta cells and alpha
 cells. These tumors are referred to as insulinomas and glucagonomas
 respectively. Like their nontransformed counterparts, insulinomas and
 glucagonomas share similarities with neuroendocrine tumors. Examples of
 other neuroendocrine tumors include small cell lung carcinoma, pituitary
 tumors, thyroid medullary carcinoma and pheochromocytoma. These cells also
 express common neuroendocrine markers such as those described in the
 preceding paragraph (Alpert et al., Cell 53:295-308, (1988) and Rindi et
 al., Virchow Archiv A Pathol Anat 419:115-129 (1991)).
 Definitive identification of a tumor type, survival statistics and
 therapeutic strategies for treating a tumor are all dependant to some
 extent on the ability of the physician to differentiate one tumor type
 from another. Polyclonal or monoclonal antibodies as well as nucleic acid
 probes can be used to screen biopsy specimens to determine the derivation
 of a particular tumor. Effective pancreatic and neuroendocrine cancer
 treatment depends on the early diagnosis and identification of tumor
 tissue. Antibodies generated from antigen obtained from purified pancreas
 cell populations or antibodies directed to known polypeptides present in
 pancreatic cells can be used to differentiate one pancreatic tumor from
 another.
 U.S. Pat. No. 4,962,048 to Kajiji et al. identified hybridoma cell lines
 producing monoclonal antibodies reactive with human pancreatic cancer
 cells. The antibodies cross-reacted with several types of pancreatic
 cancer cells and were strongly reactive with a variety of tumors derived
 from a number of different organs. The antibodies failed to react uniquely
 with one identifiable cancer or related group of cancers. None of the
 antibodies reacted well with neuroendocrine-related tumors.
 Antibodies directed to known polypeptides can be used to screen
 neuroendocrine-related tumors. Immunohistochemical staining with several
 different antibodies can be used to differentiate pancreatic, endocrine
 and neuroendocrine tumors (Bordi et al., Archiv A Pathol. Anat.
 413:387-398, 1988). However, the differentiation of tumor tissue using a
 panel of antibodies is labor intensive. Multiple antibodies are separately
 reacted with duplicate cell cultures or tissue sections to generate a
 pattern of antibody reactivity that can be compared to a control panel
 characteristic of a particular tumor type to determine the origin of a
 particular tumor (Kim et al., Cancer 66:2134-2143, 1990). The results from
 these panels can be ambiguous since antibody staining is often diffuse or
 nonspecific. The intensity of positive signals may vary between matched
 samples making positive tissue identification difficult.
 Antibodies generated from tumor cell lysates can also be used to identify a
 particular tumor type. However, antibodies generated from cell lysates are
 directed toward many different polypeptides. Some of the antibodies may be
 cell specific, but a great number of the antibodies will be directed
 toward common cellular antigens. Once antibodies are generated to cell
 lysates, intensive study is required to confirm the uniqueness of a
 particular antigen and to determine the usefulness of the antibody as a
 diagnostic or therapeutic. Thus, identifying cell specific antibody,
 obtained through immunization with a particular tumor cell lysate is not a
 particularly efficient way to identify unique cellular antigen.
 No nucleic acid probes are currently available to uniquely identify
 neuroendocrine tumor tissue. The identification of transcripts unique to
 an isolated tumor type or related group of tumors would be useful for
 nucleic acid based assays. The presence of a common transcript or protein
 among related tumor cells suggests the presence of a common regulatory
 mechanism and potentially a common therapy.
 Subtractive hybridization is a useful tool for identifying uniquely
 expressed mRNA within a given cell type. Subtractive hybridization permits
 cDNA clones to be identified that represent mRNA expressed in one cell
 population and absent in a second cell population without prior knowledge
 of the gene or the gene product. This method has been used in other
 systems to identify candidate tumor suppressor genes and proteins unique
 to colon carcinoma and hepatic cancer (Lee et al., Proc. Natl. Acad. Sci.,
 USA 88:2825-2829, 1991 and Schweinfest et al., Genet Annal. Techn. Appl.
 7:64-70, 1990).
 As outlined above, the identification of polypeptide unique to a particular
 cancer cell is important for developing diagnostic tests and therapeutic
 strategies. There is a need for diagnostic tools that permit the correct
 identification of human insulinomas and neuroendocrine tumors.
 SUMMARY OF THE INVENTION
 The present invention relates to a recombinant polynucleotide having a
 sequence comprising at least 10 sequential nucleotides or complementary
 nucleotides from the sequence identified as SEQ ID NO:1. In a preferred
 embodiment of this invention, this recombinant polynucleotide is contained
 in a vector. In one aspect of this preferred embodiment, the vector
 directs the expression of the recombinant polynucleotide sequence. The
 recombinant polynucleotide is preferably RNA or DNA.
 The invention also relates to cells containing the polynucleotide vector
 and to the translation products encoded therefrom. Isolated polypeptide
 comprising at least 6 sequential amino acids as disclosed in SEQ ID NO:2
 are also contemplated within the scope of this invention. In another
 preferred embodiment of this invention, monoclonal antibody or polyclonal
 antibody that specifically bind to SEQ ID NO:2 are prepared and isolated.
 In another preferred embodiment of the invention, a method is disclosed for
 identifying a cancer in a vertebrate comprising obtaining a biological
 sample containing nucleic acid from the vertebrate, denaturing any
 double-stranded nucleic acid present in the sample, adding polynucleotide
 having a sequence corresponding to SEQ ID NO:1 or a sequential fragment
 thereof and detecting polynucleotide hybridizing to the sample, wherein
 the detection of hybridization indicates the presence of cancer. In one
 aspect of this invention, the polynucleotide fragment is at least 10
 nucleotides in length and in another, the preferred fragment is at least
 18 nucleotides in length. In another aspect of this invention, the sample
 is a tissue section. The polynucleotide is preferably labelled for
 detection and the detecting step preferably includes measuring the amount
 of label in the sample.
 In yet another preferred embodiment of this invention, a method is provided
 for identifying a cancer in a vertebrate comprising obtaining a cell
 sample from the vertebrate, adding antibody recognizing the polypeptide
 corresponding to SEQ ID NO:2 and detecting antibody binding to the sample,
 wherein the detection of antibody binding indicates the presence of
 cancer. The method is preferably formatted as an enzyme-linked
 immunosorbant assay and in another preferred embodiment the method employs
 a Western Blot assay format.

DETAILED DESCRIPTION OF THE INVENTION
 The term recombinant polynucleotide is used herein to denote polynucleotide
 (RNA or DNA) produced through recombinant methods well known in the art of
 molecular biology.
 The term complementary nucleotides are those nucleotides that form base
 pairs with the parent nucleotide sequence. This term is also well
 recognized in the art.
 Sequential nucleotides are those nucleotides positioned in succession
 within a sequence.
 The term polypeptide is used broadly to include peptide, polypeptide and
 protein.
 A nucleic acid vector is a linear or circular, RNA or DNA molecule that
 facilitates nucleic acid manipulation or transfer. Nucleic acid vectors
 are well known in the art of molecular biology.
 The full-length sequence of a novel cDNA clone termed insulinoma-associated
 antigen (IA-1) is disclosed here as SEQ ID NO: 1. The sequence of the
 corresponding translation product is provided as SEQ ID NO:2. The IA-1
 gene is uniquely expressed in malignant neuroendocrine tissues and/or cell
 lines. Its sequence includes zinc-finger DNA-binding motifs and dibasic
 amino acid pro-hormone conversion sites. The restricted tissue
 distribution and unique sequence motifs of IA-1 suggest that IA-1 may
 function as a regulatory factor in islet cell transformation.
 IDENTIFICATION OF UNIQUE INSULINOMA PROTEIN
 The preparation of subtraction libraries requires the selection of one or
 more cell types in addition to the principle cell type of interest. Thus
 to obtain unique sequences from human insulinoma cells, cDNA from an
 insulinoma cell library was hybridized with cDNA from a human glucagonoma
 cell library to remove homologous sequences. The choice of cell line
 determines the population of unique sequences remaining after subtractive
 hybridization. Glucagonoma cells were chosen in this particular example
 because the cells are both neuroendocrine in origin and because their
 nontransformed counterpart resides proximate to beta cells in the
 pancreatic islets.
 Subtractive hybridization identified a novel transcript expressed in
 insulinoma cells. The subtraction library (ISL-53) was constructed using
 human insulinoma (beta cell tumor) and glucagonoma (alpha cell tumor) cDNA
 phagemid libraries. The novel cDNA clone, IA-1, was identified by
 differential screening. The full-length sequence (2838 bp) of the cDNA
 clone was determined by double-strand sequencing of 13 cDNA clones
 isolated from both the human insulinoma subtraction library and another
 insulinoma random-primed library.
 Library Construction
 Human insulinoma and glucagonoma tissue samples were obtained from the
 National Cancer Institute, NIH (Bethesda, Md.). Tissue type was confirmed
 by immunoperoxidase staining for insulin, glucagon, somatostatin, gastrin
 and/or pancreatic polypeptide using techniques well known in the art.
 Antibodies to insulin, glucagon, somatostatin, gastrin and pancreatic
 polypeptide are available from a number of manufacturers. For current
 source availability of these antibodies see the Linscott Directory of
 Immunological & Biological Reagents (Mill Valley, Calif.).
 There are a variety of methods known in the art for obtaining cDNA
 libraries. Phagemid cDNA libraries were prepared from glucagonoma and
 insulinoma cell RNA. Total RNA was isolated from human insulinoma and
 glucagonoma tissues (NCI, NIH) using an acid guanidinium thiocyanate
 phenol/chloroform extraction method (Chomczynski, P. et al. Anal. Biochem.
 162:156-159, 1987 which is hereby incorporated by reference).
 Poly(A).sup.+ RNA was purified by passing the sample twice through
 oligo-dT cellulose columns. Phagemid cDNA libraries were constructed using
 Invitrogen's Librarian II system (San Diego, Calif.) following
 manufacturer's instructions. Aliquots of Poly(A).sup.+ RNA were primed
 with oligo-dT (Invitrogen, San Diego, Calif.). Double-stranded
 linker-ligated cDNAs generated from the Librarian II system greater than
 750 bp were selected from agarose gels for further study. The cDNA was
 electroeluted and ligated with pcDNAII (Invitrogen) and transformed into
 competent E. coli cells and amplified on LB plates using ampicillin.
 Poly(A).sup.+ RNA from human insulinoma was also random-primed (see
 Example 5) using a cDNA human insulinoma library (RP-IL) in a
 .lambda.ZAPII phage vector system (Stratagene, La Jolla, Calif.).
 Subtraction libraries can be generated using commercially available kits
 such as those obtained from Invitrogen (San Diego, Calif.). Similarly,
 libraries may be prepared using methods known to those with skill in the
 art such as those described by Schweinfest et al. (supra.) or Hara et al.
 (Nucl. Acids Res., 19:7097-7104, 1991). As one example of a method for
 generating an insulinoma-specific subtraction library, R408 helper phage
 (Invitrogen) was used to infect insulinoma and glucagonoma libraries to
 generate single-stranded phagemid DNA. Phage produced from these
 infections were harvested and the single-strand DNA was extracted from the
 phage and purified using techniques well known to those with skill in the
 art and detailed in the Invitrogen subtraction library kit instructions.
 Glucagonoma phagemid single-strand DNA was photobiotinylated using
 photobiotin acetate. Single-stranded phagemid DNA derived from the
 insulinoma library was coprecipitated with an excess of photobiotinylated
 glucagonoma single-strand phagemid DNA. The DNA was resuspended, denatured
 and cooled to permit hybridization. Following hybridization, streptavidin
 was used to separate out both single-strand glucagonoma DNA and
 glucagonoma DNA bound to insulinoma DNA. The remaining single-strand DNA
 was converted to double strand DNA using methods known in the art and the
 reaction mixture was used to transform competent E. coli (see Example 2).
 Other subtractive hybridization methods that could similarly be used,
 include, but are not limited to, methods employing oligonucleotide
 labelled beads and avidin-agarose extraction techniques. Transformed
 colonies were selectively plated onto agar and colonies were picked and
 stored for future analysis.
 Screening of the Subtraction Library
 Plasmid DNA was initially isolated from individual cDNA clones obtained
 from the insulinoma-glucagonoma subtraction library (ISL-153) and screened
 against probes derived from insulinoma, glucagonoma and HeLa cells. Clones
 reacting with insulinoma probes, but not glucagonoma and HeLa cell probes
 were selected for further study. Example 3 provides an exemplary initial
 screening technique. Plasmid DNA was isolated using the alkaline lysis
 mini-preparation technique, well known in the art. The purified DNA was
 digested with HindIII and XhoI restriction endonucleases and separated by
 agarose gel electrophoresis. The DNA was transferred to Nytran paper
 (Schleicher & Schuell, Keene, N.H.) and replica blots were screened
 separately using .sup.32 P-end-labeled mRNA isolated from insulinoma,
 glucagonoma and HeLa cells.
 Homology to GenBank Database Sequences
 The selected clones were partially sequenced for approximately 200 bp from
 both ends of the vector using reverse and forward primers that bind to
 .lambda. ZAP II DNA (United States Biochemical, Cleveland, Ohio). These
 sequences were compared with sequences available in the GenBank DNA
 database. Three of the cDNA clones revealed sequence similarity (&gt;95%)
 with trypsin (Emi, et al. Gene 41:305-310, 1986), chymotrypsin (Tomita, et
 al., Biochem. Biophys. Res. Comm. 158: 569-575, 1989) and pancreatic
 protease E (Shirasu, et al. J. Biochem. 104: 259-264, 1988) and may be
 derived from contaminating acinar tissue associated with the original
 insulinoma. A fourth clone showed similarity (&gt;99%) with the islet
 regenerating protein (reg) previously described by Terazono, et al. J.
 Biol. Chem. 263:2111-2114 (1988). A fifth clone had a high sequence
 similarity (&gt;99%) with a GTP-binding protein (alpha subunit) (Bray, et
 al., Proc. Natl. Acad. Sci. USA 83: 8893-8897, 1986) and a sixth clone had
 high homology (&gt;97%) to pancreastatin hormone precursor-chromogranin A
 (Helman, et al., J. Biol. Chem. 263: 11559-11563, 1988). The seventh
 clone, IA-1-34 (see FIG. 1), and the eighth clone, IA-2, had no
 identifiable sequence homology with sequences in the GenBank database.
 Northern analysis, using each of the first six clones as probes revealed
 message size consistent with that previously reported for each of these
 proteins as described in Example 4. The seventh clone, IA-1, and the
 eighth clone, IA-2, based on sequence comparison, proved to be novel.
 Northern analysis revealed mRNAs of 3.0 Kb and 3.8 Kb, respectively.
 Transcript size and homologies are summarized in Table I.
 When the predicted 510 amino acid sequence of IA-1 (see FIGS. 2A-2C) was
 matched to protein sequences in the database, IA-1 had a 20 to 30%
 homology with members of the zinc-finger protein family. The similarity of
 the protein sequence was limited to the consensus sequences within the
 zinc-finger motif. No similarities were found in the flanking regions.
 TABLE I
 Isolation and identification of cDNA clones
 from a human substraction library
 cDNA clone Message Size* Identification.sup.#
 1 0.8 kb Trypsin
 2 1.1 kb Chymotrypsin
 3 0.9 kb Elastase
 4 0.8 kb Regenerating protein
 (reg)
 5 2.5 kb GTP-binding protein
 (alpha subunit)
 6 2.2 kb Chromogranin A
 7 3.0 kb IA-1
 8 4.0 kb IA-2
 *Message size of each clone was estimated from Northern analyses.
 .sup.# Each clone was partial sequenced approximately 200 bps from both
 ends of pcDNAII vector by using reverse and forward primers and the
 sequence was matched to the GenBank database (Pearson et al., Proc. Natl.
 Acad. Sci. USA 85:244-2448, 1988).
 Generating the Full-Length cDNA Clone of IA-1
 The initial clone, IA-1-34, isolated from the subtraction library (ISL-153)
 contained a 1508 bp sequence upstream from the poly(A) tail (FIG. 1B). To
 further analyze full length IA-1, a random-primed .lambda.ZAPII cDNA
 library (Stratagene) from human insulinoma tissues was constructed
 (RP-IL). IA-1-34 insert DNA was used as a probe to screen the insulinoma
 .lambda.ZAPII library (RP-IL). Additional positive clones with insert
 sizes ranging from 0.6 to 2.8 kb were isolated (see Example 5 and FIG.
 1C). The longest clone, IA-1-18, extending from 4 to 2819 bp, was used as
 a probe for later tissue distribution studies (Example 6). Ten cDNA clones
 isolated from the secondary screening of the random-primed library
 contained regions overlapping with the IA-1-34 insert. Two addition clones
 were isolated using IA-1-18 as a probe. cDNA clones of various lengths
 were subjected to double-strand sequencing using internal primers that
 primed from both ends of the insert. These primers are provided as SEQ ID
 NOS: 3-27 and their positions are illustrated in FIG. 2. The sequencing
 strategy is illustrated in FIG. 1D and detailed in Example 5.
 The full-length cDNA sequence and the deduced protein sequence are shown in
 FIGS. 2A-2C. The cDNA sequence corresponds to an mRNA containing a 147 bp
 5'-untranslated region and an 1150 bp 3'-untranslated region containing
 two polyadenylation consensus sequences, AATAAA, located at positions 2127
 and 2807. The proposed significance of the AATAAA sequences is described
 by Birnstiel, et al., Cell 41: 349-359 (1985). The first ATG, in nucleic
 acid position 66, is followed by a termination codon, TGA, in position 81.
 The next ATG codon at position 147 contains a long open reading frame of
 1530 nucleotides and is flanked by sequences that fulfill the Kozak
 translation consensus sequence, GCCA/GCC[ATG]G (Kozak, M. Nucl. Acids Res.
 15: 8125-8248, 1987). There are also seven ATTTA sequences positioned
 between the two polyadenylation signals.
 Structural Features of the Deduced Amino Acid Sequence
 Certain predictions can be made from the nucleic acid and protein sequence
 of the IA-1 clone. While these predictions are not absolute, they are
 based on consensus data obtained from other protein sequence/function
 studies and help those with skill in the art to further characterize IA-1
 polypeptide.
 The large open reading frame of IA-1 indicates that the protein contains
 510 amino acids and has a deduced pI value of 9.1 and an unmodified
 molecular mass of 52,923 daltons.
 The amino acid sequence can be divided into two domains based upon distinct
 chemical features associated with each domain (FIG. 3). The N-terminal
 domain (1-250 a.a.) has a high content of proline (18%), glycine (12.8%),
 and alanine (16%). The proline-enriched (20 to 30%) domain of DNA-binding
 proteins has been found in many mammalian transcription factors, including
 enhancer-binding protein, AP-2 (Williams, T. et al. Genes and Development
 2:1557-1569, 1988), Jun (Ryseck et al., Nature 334:535-537, 1988),
 lymphocyte-specific factor, OCT-2 (Muller, M. M. et al. Nature 336:
 544-551, 1988), and serum response factor, SRF (Norman, C. et al. Cell
 55:989-1003, 1988) and represents one type of activation domain (Mitchell,
 P. J. et al. Science 245: 371-378, 1989).
 In addition to a proline rich domain, there are four classical pro-hormone
 dibasic conversion sites at positions 8-9, 11-12, 221-222, and 227-228
 with an amidation signal sequence, Pro-Gly-Lys-Arg, at position 198-201
 (Kreil, G. (1986) Protein Compartmentalization, pp. 61-70 Springer-Verlag,
 Inc., New York.). Peptide hormone precursors of insulin, glucagon,
 pancreatic polypeptide and somatostatin contain dibasic amino acid
 cleavage sites that are associated with protein processing. In the nervous
 and endocrine system many bioactive peptides possess a C-terminal
 .alpha.-amide group. The .alpha.-amide moiety is generated from enzyme
 cleavage at the amidation signal sequence with the conversion of peptides
 with the structure --X-Gly into the form --X-NH1.sub.2. The presence of
 the .alpha.-amide group was shown to be important for pancreatic
 polypeptide (Schwartz, T. W. Gastroenterology 85: 1411-1425, 1983) as well
 as other amidated peptides. (Tatemoto, K. et al. J. Biol. Chem.
 263:2111-2114, 1978). The presence of potential dibasic pro-hormone
 processing sites and the amidation signal sequence in the putative
 activation domain demonstrated that IA-1 is a novel cDNA clone without
 similarities to other DNA-binding proteins.
 The C-terminal domain (251-510 a.a.) contains five putative "zinc-finger"
 DNA-binding motifs of the form X.sub.3 -Cys-X.sub.2-4 -Cys-X.sub.12
 -His-X3-4-His-X.sub.4, described as a consensus sequence for members of
 the Cys.sub.2 -His.sub.2 class (Mitchell et al., supra.). FIG. 3
 illustrates a zinc-finger motif in the middle of the carboxyl-domain of
 IA-1 located between two tandem repeated zinc-finger motifs spaced 45/46
 amino acids from each side. The first zinc-finger sequence, positioned at
 amino acids 266-293, lacks the last histidine residue at position 289. A
 comparison of IA-1 zinc-finger motifs with the consensus sequence of
 various zinc-finger DNA-binding proteins is shown in FIG. 4 (Kadonaga, J.
 T. et al. Cell 51:1079-1090 (1987); Sukhatme, V. P. et al. Cell 53:37-43
 (1988); Brown, R. S. et al. Nature 324:215 (1986); Rosenberg, U. B., et
 al. Nature 319:336-339 (1986)).
 The conserved residues, Cys, His, Phe and Leu, are identical to other known
 proteins containing zinc-finger domains. However, the residues positioned
 between each zinc-finger motif are different from published sequences.
 Zinc-finger motifs were first discovered by Miller, J. et al. (EMBO J.
 4:1609-1614, 1985) in Zenopus transcription factor IIIA. Subsequently,
 other proteins with zinc-finger domains were shown to be involved in
 DNA-binding and in many aspects of eukaryotic gene regulation, such as
 differentiation and growth signals, proto-oncogenes and transcription
 factors. The IA-1 cDNA clone contains five zinc-finger motifs and a
 proline-rich activation domain which resembles other DNA-binding proteins
 and may be responsible for recognizing and binding IA-1 to DNA. The
 organization of the zinc-finger motifs and activation domains are unique.
 The nucleotide sequence contains an 1150 bp 3'-untranslated region with
 seven ATTTA sequences located between two polyadenylation signals, AATAAA.
 The presence of pro-hormone and amidation sequences in the amino-terminal
 domain strongly suggests that the ATTTA sequence element may serve as a
 recognition signal for the specific degradation of mRNA as reported for
 proto-oncogenes and inflammatory mediators such as lymphokines and
 cytokines (Shaw, G. et al. Cell 46: 659-667, 1986 and Caput, et al., Proc.
 Natl. Acad. Sci. USA 83:1670-1674, 1986).
 Tissue Expression of IA-1 Gene
 Fragments of IA-1 were used as probes to determine the tissue specificity
 of IA-1 gene expression. RNA isolated from five human insulinomas and a
 variety of other human, mouse and rat cell lines were separated by 1%
 agarose formaldehyde gel electrophoresis. The RNA was
 capillary-transferred to Nytran paper and probed with the cDNA clone
 IA-1-18 corresponding to cDNA nucleotide positions 4 through 2819 (Table
 II and FIG. 5).
 Normal human tissues were obtained from the National Disease Research
 Interchange (NDRI, Philadelphia, Pa.). Cell lines, HPAF-2 (pancreatic
 adenocarcinoma), BT-20 (breast carcinoma), SKMEL (melanoma), DM-6
 (melanoma), and HeLa (ovarian carcinoma) were obtained from Dr. R. S.
 Metzgar (Duke University, N.C.); JAR (choriocarcinoma), BeWo
 (choriocarcinoma), SK-N-SH and SK-N-MC (neuroblastoma), NCI-H69 (small
 cell lung carcinoma), U-78-MG (glioblastoma), SW579 (thyroid carcinoma),
 AtT-20/D16v-F2 (mouse pituitary tumor), PC-12 (rat pheochromocytoma), GH-3
 (rat pituitary tumor), 6-23, clone 6 (rat medullary thyroid carcinoma),
 Y-1 (mouse adrenal cortex tumor), and F-9 (mouse embryonal carcinoma) were
 obtained from the American Type Culture Collection (Rockville, Md.).
 .alpha.-TC1 (mouse glucagonoma) and .beta.-TC1 (mouse insulinoma) were
 kindly provided by Dr. E. H. Leiter (Bar Harbor, Me.). Tumor cell lines
 were cultured in modified Eagle's medium supplemented with 10% fetal calf
 serum in 5% CO.sub.2 at 37.degree. C. or according to supplier's
 instructions.
 Mouse islets were isolated from Balb/c pancreas as described by Brunstedt,
 J. et al., Methods in Diabetes Research 1(C): 245-258 (1985) which is
 hereby incorporated by reference. Pancreases from twenty mice were
 digested with collagenase P (Boehringer Mannheim Biochemicals,
 Indianapolis, Ind.) and islets were isolated by Percoll gradient
 (Pharmacia, Uppsala, Sweden) separation. The enriched islets were then
 extracted for total RNA using methods described in Example 1.
 Five of five human insulinomas and a small cell lung carcinoma (NCI-H69)
 expressed IA-1 as a 3.0 kb message (FIG. 5A). A summary of the tissue
 expression work is provided in Table II. Normal tissues express little of
 this gene. Normal human pancreas, testes, lymph node, brain, lung, liver,
 stomach, spleen, thyroid, pituitary, kidney, colon were negative by
 Northern analysis. RNA preparations from enriched normal murine islet
 cells that contained concentrated quantities of beta cell RNA had a
 significant increase in insulin-specific message as compared to total
 pancreas RNA. However, these beta cell enriched RNA preparations were
 still negative for IA-1.
 IA-1 was present in several neuroendocrine tumors including
 pheochromocytoma, medullary thyroid carcinoma, insulinoma, pituitary tumor
 and small cell lung carcinoma. The cDNA probe also recognized a 3.0 kb
 message from insulinoma cell lines of mouse (.beta.-TC1), rat (RIN), and
 hamster (HIT). The message was detected in cell lines from mouse pituitary
 tumor (AtT-20/D16v-F2), rat pheochromocytoma (PC-12), rat pituitary tumor
 (GH-3) and rat medullary thyroid carcinoma (6-23) (Table II). Other cell
 lines such as mouse adrenal cortex tumor (Y-1); mouse embryonal carcinoma
 (F-9), mouse NIH-3T3 and rat testicular tumor (LC-540) were negative on
 Northern analysis. The restricted tissue distribution for IA-1 indicated
 that it recognized a protein specifically expressed by human insulinoma
 tissues, murine insulinoma cell lines, and other neuroendocrine tumors.

IA-1 EXPRESSION IN HUMAN, MOUSE, AND RAT TUMORS.sup.a
 Human Mouse Rat
 Positive Insulinoma tissue.sup.b Pituitary tumor Pheochromocytoma
 AtT-20/D1 6v-F-2 adrenal medullary, PC-12
 Small cell lung carcinoma
 NCI-H69 Insulinoma Pituitary tumor
 .beta.-TC-1 GH-3
 Insulinoma
 RIN
 Glucagonoma
 .alpha.-TC-1.sup.c
 Medullary thyroid
 carcinoma
 6-23
 Negative Choriocarcinoma Adrenal tumor
 JAR (Cortex) Testicular tumor
 BeWo Y-1 Leydig cell, LC-540
 Embryonal carcinoma
 F-9
 Neuroblastoma
 SK-N-SH Fibroblast
 SK-N-MC NIH-3T3
 Giloblastoma
 U-87-MG
 Thyroid carcinoma
 SW-579
 Breast carcinoma
 BT-20
 Pancreatic carcinoma
 HPAF-2
 Ovarian carcinoma
 HeLa
 Melanoma
 SKMEL
 DM-6
 .sup.a This is a summary table from northern analysis data
 .sup.b Five human insulinoma tissues were tested
 .sup.c Very weak signal was detected with a longer exposure
 Use of IA-1 as a Tumor Marker
 IA-1 gene sequences or fragments of the IA-1 gene sequence are useful for
 identifying and verifying the origin of tumors. The cloned fragments of
 IA-1 illustrated in FIG. 1C, full length IA-1 or fragments thereof can be
 labelled with radioactive isotopes, biotin, fluorescent tags or the like
 to generate markers for the identification of insulinomas or other tumors
 of neuroendocrine origin.
 In a study to explore the diagnostic application of IA-1, the IA-1-34
 insert was labelled and used as a probe to perform Northern analyses (see
 Example 7). RNA was isolated from biopsies of human small cell lung
 carcinomas. IA-1 mRNA was present in 22 of 22 human small cell lung
 carcinoma cell lines and 8 of 8 human small cell lung cancer biopsies. No
 IA-1 mRNA was identified in 15 of 15 non-small cell lung carcinomas. The
 unique tissue distribution of this gene for transformed neuroendocrine
 tissue makes it an ideal candidate for both a nucleic acid and protein
 tumor marker in clinical, diagnostic and research applications.
 It is further contemplated within the scope of this invention that nucleic
 acid fragments, polypeptide and polypeptide fragments encoded therefrom,
 or antibody reactive with polypeptide or polypeptide fragments can be used
 as part of a panel of tumor markers. Diagnostic research related kits and
 assays employing recombinant gene fragments as probes are well known in
 the art. It is anticipated that IA-1 cDNA, RNA generated therefrom and DNA
 or RNA fragments of IA-1 can be readily adapted for assay formats for
 diagnostic or research purposes. Assay formats employing nucleic acid
 probes include but are not limited to in-situ hybridization of biopsy and
 tissue sections, Polymerase chain reaction (PCR) related assays, Dot-blot
 manifold assays or the like. These assays can provide a rapid and
 reproducible means for identifying pancreas or neuroendocrine tumors. Thus
 recombinant nucleic acid sequences corresponding to IA-1 or sequential
 nucleotide fragments thereof can be used to positively identify a
 particular tumor.
 A method for identifying the presence of neuroendocrine related cancer from
 a patient involves obtaining a sample from a patient that contains nucleic
 acid. The sample may be a purified nucleic acid, cell lysate, a tissue
 section, a blood sample or the like. Double-stranded nucleic acid or
 double stranded regions of a single-stranded nucleic acid are preferably
 denatured by heat, sodium hydroxide or other methods known to these with
 skill in the art. Following denaturation, polynucleotide of at least 10
 nucleotides or more preferably at least 18 nucleotides in length,
 corresponding or complementary to SEQ ID NO: 1 is added to the sample
 under conditions that promote hybridization (see Example 4 and Davis et
 al. (1986) Basic Methods in Molecular Biology. Elsevier Press). Unique
 fragments of SEQ ID NO:1 of at least 10 nucleotides in length, preferably
 at least 15, 18, or 20 nucleotides in length, are contemplated within the
 scope of this invention.
 Polypeptide fragments generated from the amino acid sequence provided in
 FIG. 2 and in SEQ ID NO:2, polypeptide generated by recombinant expression
 vectors containing IA-1, or in vitro translated polypeptide can be used to
 generate antibody reactive with IA-1 protein both in vitro and in vivo.
 These polypeptide fragments are preferably unique fragments at least 6,
 10, or 15 amino acids in length. Methods for generating polypeptide or
 protein fragments by chemical synthesis, recombinant vector expression or
 in vitro translation are well known in the art and would not require undue
 experimentation. Once peptide or polypeptide fragments are obtained, the
 protein may be used to immunize animals for the generation of polyclonal
 and monoclonal antibody in accordance with standard techniques. Example 8
 provides one example of a method for the preparation of monoclonal
 antibody reactive with IA-1 protein. Other methods for generating
 monoclonal antibodies and for genetically modifying these antibodies are
 well known in the art. It is additionally contemplated within the scope of
 this invention that antibody directed to IA-1 could also be obtained using
 the polymerase chain reaction to obtain variable antibody domain sequences
 to then generate a library of antigen combining sites such as the
 recombinatorial phage libraries described by Huse et al., Science 246:1275
 (1989).
 Monoclonal or polyclonal antibody generated to recombinant protein or
 peptide fragments of IA-1 using techniques well known to those with skill
 in the art are used to screen tissue samples by either Western Blot assay,
 immunostaining of cell lines or tumor tissues, ELISA or the like.
 Exemplary cell lines that may be used to facilitate antibody screening are
 provided in Table II. Testing over a range of cell types is used to ensure
 the cell specificity of the antibody. Antibody reactive with IA-1 can then
 be employed in any number of antibody related assays. Antibody-related
 assays are well known in the art and include, but are not limited to
 Western Blot assays, ELISA format assays, immunohistochemical assays and
 competitive protein assays such as radio-immuno assays or the like. These
 formats may be useful as research assays for assessing the functional
 characteristics of IA-1 and as diagnostic assays for rapid multi-sample
 analysis.
 A general method contemplated within the scope of this invention for
 identifying a cancer in a vertebrate includes obtaining a cell sample from
 the vertebrate as a biopsy, tissue section or the like. The sample may be
 fresh, fixed or frozen and there are methods well known in the art that
 apply antibody to samples treated in a variety of fixatives. It is further
 contemplated that samples may be processed in NP40, SDS, other suitable
 detergents or homogenized to disrupt cell integrity. Antibody specifically
 binding to the polypeptide corresponding to SEQ ID NO: 2 is added
 according to procedures and methods compatible with immunohistochemical
 staining techniques or antibody-utilizing assays such as ELISA's or
 Western blot assays.
 It is also contemplated that antibody or nucleic acid sequences as
 described herein can additionally be used to rule out a particular type of
 cancer. Thus these methods are useful for generally identifying cancer in
 a vertebrate.
 It is further contemplated that antibody directed to IA-1 can be used to
 substantially purify or separate IA-1 from surrounding native protein.
 Techniques known in the art such as affinity-chromatography or other
 chromatographic methods that do not employ antibody could be used to
 isolated native IA-1.
 With respect to the DNA and RNA sequences and fragments of the present
 invention, and also with respect to the proteins and peptides, these
 materials may advantageously be provided in a non-naturally occurring
 form. Thus, they may be substantially purified, i.e., they have a degree
 of purity significantly greater than the naturally occurring form. For
 example, concentrations of these materials of at least 0.01 .mu.g/g,
 preferably at least 0.1 .mu.g/g, and more preferably 1 or 10 .mu.g/g
 typically satisfy this definition. Alternatively, the materials may be
 provided in an isolated form; that is, they are substantially isolated
 from or enriched in comparison to the proteins, peptides, or
 polynucleotides with which they are associated in the natural state.
 Materials that are, for example, 100, 1000, or 10,000 times more
 concentrated than in the natural state are considered to be both isolated
 and purified for purposes of the present invention.
 Particular embodiments of the invention will be discussed in detail. These
 embodiments are intended to illustrate and not limit the scope of the
 invention.
 EXAMPLE I
 Library Construction
 To construct the phagemid cDNA libraries, total RNA was isolated from human
 insulinoma and glucagonoma tissues by acid guanidinium thiocyanate
 phenol/chloroform extraction method (Chomoczynski et al., supra.).
 Poly(A).sup.+ RNA was purified by twice passing the RNA aliquots through
 oligo-dT cellulose columns. Phagemid cDNA libraries were constructed using
 Invitrogen's Librarian II system (San Diego, Calif.). Poly(A).sup.+ RNA
 (10 .mu.g) was primed with oligo-dT and the double-stranded linker-ligated
 cDNAs were size-selected over 750 bp. The sized, electroeluted cDNAs for
 each library were ligated to 1.5 .mu.g of prepared PCDNAII. The
 vector-ligated cDNA was then transformed into 4.0 ml of high efficiency
 competent MC1061/P3 E. coli cells (Invitrogen, San Diego, Calif.) and
 amplified on LB agar plates containing ampicillin (50 .mu.g/ml). The
 titers of the original libraries were 1.0.times.10.sup.6 colonies for the
 insulinoma library and 1.5.times.10.sup.6 colonies for the glucagonoma
 library.
 EXAMPLE 2
 Subtraction Library
 The subtraction cDNA library was constructed using a kit supplied by
 Invitrogen (San Diego, Calif.). R408 helper phage was used to infect the
 insulinoma and glucagonoma libraries to generate single-stranded phagemid
 DNA. An aliquot (1 ml., equivalent to approx. 10.sup.10 colonies/ml.) of
 the amplified cDNA library was diluted with 20 ml of LB medium containing
 ampicillin (50 .mu.g/ml). Each diluted library was incubated with shaking
 for one hour at 37.degree. C. Each culture was inoculated separately with
 1.times.10.sup.6 pfu of R408 helper phage. After infection, each culture
 was incubated for an additional 30 minutes, then diluted into 100 ml of LB
 medium containing ampicillin (50 .mu.g/ml) and incubated overnight at
 37.degree. C. with shaking. The cell pellet was discarded and 10 .mu.l of
 10 mg/ml of RNase A was added to this supernatant (33 ml) along with 5 ml
 of 8M ammonium acetate and 5 ml of 40% Polyethylene glycol (wt/wt) on ice
 for three hours. Single-strand DNA was extracted from each library three
 times with phenol/chloroform and examined on a neutral agarose gel.
 Photo-biotinylation of the glucagonoma phagemid single-strand DNA was
 performed according to the manufacturer's procedure (Invitrogen).
 Single-strand glucagonoma phagemid DNA (20 .mu.g) was added to 30 .mu.g of
 photobiotin acetate in a 1.5 ml screw cap tube, on ice, 10 cm below a
 sunlamp (General electric #RSM, 275W). The tube was heated for 15 minutes,
 followed by three to four extractions of the solution with water-saturated
 2-butanol.
 Subtractive hybridization was performed using ten times molar excess of
 photobiotinylated glucagonoma single-strand phagemid DNA to remove all
 sequences present in the insulinoma phagemid DNA library that were
 substantially homologous with the glucagonoma library. Biotinylated DNA
 was co-precipitated in ethanol with non-biotinylated DNA, and dissolved in
 10 .mu.l of 2.times. hybridization buffer (kit supplied). The hybridized
 mixture was heated to 100.degree. C. for one minute and cooled to
 68.degree. C. for 16 to 20 hours. After hybridization the mixture was
 placed in a 55.degree. C. water bath for five minutes, then diluted with
 30 .mu.l of 10 mM Hepes/EDTA buffer. 10 .mu.l of 1 mg/ml streptavidin was
 added followed by extraction with phenol/chloroform. The final subtracted
 single-strand DNA was converted to double-strand DNA using T7 primer and
 reverse transcriptase. Aliquots of the reaction mixture were used to
 transform 100 .mu.l of competent E. coli cells (DH1.alpha.F'). Cells were
 plated onto LB agar plates containing 50 .mu.g/ml ampicillin. The
 individual colonies were picked and stored in 96-well microtiter plates at
 -80.degree. C.
 EXAMPLE 3
 Screening the Subtraction Library
 Mini-preparations of plasmid DNA (triplicates) from the individual
 insulinoma-glucagonoma subtraction library cDNA clones were prepared using
 the Plasmid Kit (Qiagen, Chatsworth, Calif.). Purified DNA was digested
 with HindIII and XhoI restriction endonucleases (BRL, Bethesda, Md.).
 Digested DNA was separated by 1% agarose gel electrophoresis and
 transferred to Nytran paper (Schleicher & Schuell, Keene, N.H.) for
 Southern blot analysis using techniques well known in the art (Davis et
 al., 1986. Basic Methods in Molecular Biology. Elsevier Press). Triplicate
 blots were pre-hybridized at 50.degree. C. with 40% formamide, 5.times.
 SSC, 10 .mu.g/ml sheared salmon sperm DNA, 6.times. Denhardt's solution
 and hybridized with an equal specificity of end-labeled mRNA probe
 (10.sup.6 cpm/ml).
 The mRNA probes from insulinoma, glucagonoma and HeLa cells were prepared
 as follows: 3 .mu.g of mRNA were dissolved in 10 .mu.l of a solution
 containing 0.05 M sodium bicarbonate, pH 9.2, and heated to 85.degree. C.
 for five minutes. The reaction mixtures were neutralized with 10 .mu.l of
 1.0 M Tris, pH 7.5, and precipitated with 20 .mu.l of 4 M ammonium acetate
 and 80 .mu.l of ethanol. The partially degraded mRNA was end labelled
 using T4 polynucleotide kinase (Pharmacia, Piscataway, N.J.) as described
 by Maxam, A. et al. Proc. Natl. Acad. Sci. USA 74: 560-564 (1977), hereby
 incorporated by reference.
 EXAMPLE 4
 Northern Analysis of Unique Clones
 Northern analysis was performed using total RNA purified by the acid
 guanidinium thiocyanate method. RNA (20 .mu.g) was fractionated by 1%
 agarose/formaldehyde gel electrophoresis and transferred to Nytran via
 capillary blotting (Davis et al., Basic Methods in Molecular Biology.
 (1986) Elsevier Press. pp.143-146). Probe hybridization of Northern blots
 was performed at 50.degree. C. for 18 hours with 40% formamide, 5.times.
 SSC, 10 .mu.g/ml sheared salmon sperm DNA, 6.times. Denhardt's solution,
 and 10.sup.6 cpm/ml labeled probe. The cDNA insert was removed from vector
 pcDNAII with the appropriate restriction enzymes, Hind III and XhoI
 (Stratagene, La Jolla, Calif.), separated by agarose gel electrophoresis
 and purified from the agarose using the Geneclean II kit (Bio 101,
 Boulder, Colo.). Purified insert (200 ng) was labeled with .sup.32 P-dCTP
 (Amersham Corp, Arlington Heights, Ill.), using a commercially available
 random-primed labeling kit (BRL, Bethesda, Md.) and purified by passing
 the sample over a NICK-column (Pharmacia Piscataway, N.J.).
 EXAMPLE 5
 IA-I Gene Sequence
 IA-1-34 was used as a probe to screen 500,000 plaques obtained from a
 random-primed .lambda.ZAPII cDNA library (RP-IL) derived from human
 insulinoma tissue. Human insulinoma RNA was isolated as described in
 Example 1. cDNA derived from poly(A).sup.+ RNA was prepared according to
 kit directions for .lambda.ZAPII library construction (Stratagene). The
 library yielded approximately 1.2.times.10.sup.6 pfu/ml. Following kit
 instructions, the clones from the .lambda.ZAPII library (RP-IL) that
 hybridized to IA-1-34 were converted into pBluescript SK.sup.- using
 helper phage for DNA sequencing. Plasmid DNA obtained from the clones was
 used for double-strand DNA sequencing using Sequenase T4 DNA polymerase
 under conditions recommended by the supplier (U.S. Biochemical Corp.,
 Cleveland, Ohio). Internal sense and antisense strand primers were
 synthesized by Bio-synthesis, Inc. (Denton, Tex.) and are diagrammed in
 FIGS. 2A-2C and listed as SEQ ID NOS: 3-27. The full-length cDNA
 nucleotide sequence of IA-1 was obtained by sequencing the 13 independent
 overlapping clones isolated from the subtraction library and the
 random-primed .lambda.ZAPII library. DNA sequences were analyzed using a
 Model VAX 750 (Digital Electronics Corporation computer) and the GCG
 Sequence Analysis software package (Devereux, J. et al. Nucleic Acids Res.
 12: 387-393, 1984, hereby incorporated by reference). The current FASTA
 database was used for searching both nucleic acid and protein sequence
 similarities (Pearson, W. R. et al. Proc. Natl. Acad. Sci. USA 85:
 2444-2448, 1988 hereby incorporated by reference).
 EXAMPLE 6
 IA-1 Gene Expression in Tissue
 RNA was isolated from five human insulinomas and a variety of other human,
 mouse and rat cell lines as described supra. using techniques described in
 Example 1. Total RNA was separated by 1% agarose formaldehyde gel
 electrophoresis using techniques described in Example 4. Total RNA (20
 .mu.g) was isolated from five human insulinoma tissues and loaded onto an
 agarose formaldehyde gel (FIG. 5A, lanes 1-5). Equal amounts of RNA
 obtained from the following human cell lines were also loaded onto the
 gel: small cell lung carcinoma: NCI-H69 (lane 6); neuroblastoma: SK-N-SH
 (Lane 7), thyroid carcinoma: SW579 (Lane 8), choriocarcinoma: JAR (Lane
 9), glioblastoma: U-87 MG (Lane 10), breast carcinoma: BT-20 (Lane 11),
 and pancreatic adenocarcinoma: HPAF-2 (Lane 12). The RNA was
 capillary-transferred to Nytran paper and probed with the .sup.32
 P-labeled IA-1-18. Hybridization conditions are provided in Example 4.
 FIG. 5A illustrates the results obtained from a Northern blot containing
 total RNA from insulinoma tissues as compared with other transformed cell
 lines. Human insulinoma tissues and small cell lung carcinoma cells were
 positive for IA-1 RNA. Results are summarized in Table II. FIG. 1B is a
 Northern blot of RNA obtained from murine cell lines. Mouse fibroblast:
 NIH-3T3 RNA was loaded into Lane 1, mouse glucagonoma: alpha TC-1 (Lane
 2), mouse insulinoma; beta TC-1 (Lane 3), rat insulinoma: RIN (Lane 4),
 and hamster insulinoma: HIT (Lane 5). The gel was transferred to Nytran
 and probed with labelled IA-1-18. A portion of the ethidium bromide
 stained gel containing the 18S ribosomal RNAs is shown at the bottom of
 FIG. 5. The cDNA probe strongly cross-hybridized with insulinoma cell
 lines of mouse, rat and hamster. The mouse glucagonoma cell was faintly
 positive only after extended exposure times.
 EXAMPLE 7
 IA-1 Gene Fragments as Probes for Neuroendocrine Tumors
 RNA was isolated from human small cell lung cancer biopsies and a variety
 of human lung cancer cell lines using techniques described in Example 1.
 Total RNA was separated for Northern analysis using 1%
 agarose/formaldehyde gel electrophoresis applying techniques described in
 Example 4. Results indicated that 22 of 22 human small cell lung carcinoma
 cell lines and 8 of 8 human small cell lung cancer biopsies expressed IA-1
 mRNA whereas 15 of 15 non-small cell lung carcinoma cell lines and other
 human tumor cell lines (as shown in Table II) expressed little of IA-1
 associated transcripts. This tissue screening data strongly demonstrated
 the use of IA-1 and fragments thereof to monitor tumor progression in
 small cell lung cancer in particular and in neuroendocrine tumors in
 general.
 EXAMPLE 8
 Preparation of Antibody to IA-1 Protein
 Peptide fragments of fifteen amino acids or greater and substantially
 purified IA-1 are injected separately into mice in incomplete Freund's
 adjuvant using techniques well known in the art (Madsen et al.,
 Endocrinology 113:2135-2144, 1983 and Beck et al., Exp. Clin. Endocrinol.
 93:255-260, 1989). Spleens are surgically removed from anesthetized IA-1
 sera positive mice and a spleen cell suspension is prepared in Dulbecco's
 Modified Eagle Medium (DMEM). Splenocytes are minced and fused with log
 phase Sp2/0 mouse myeloma cells using 50% (vol/vol) polyethylene glycol
 1500 in DMEM as described by Madsen et al., supra. Cells are resuspended
 in HAT medium composed of complete RPMI with 10.sup.-4 M hypoxanthine,
 4.times.10.sup.-7 aminopterin, 1.6.times.10.sup.-5 M thymidine, and
 aliquoted at a concentration of 1.times.10.sup.5 cells/well into 96-well
 flat bottom microculture plates (Costar, Cambridge, Mass.) containing
 3-4.times.10.sup.4 1-day old feeder cells per well (Contreas et al.,
 Pancreas 5:540-547, 1990).
 After 8-16 days, cultures are screened by ELISA for reactivity to IA-1
 peptide fragments or protein. ELISA techniques are well known in the art.
 Following the initial screening with peptide or protein, positive samples
 are tested on cell lines described in Example 6. Antibody reacting
 consistently with Northern Blot data from Example 6 is further tested on
 frozen tissue sections from patients with neuroendocrine tumors using
 indirect immunofluorescence and/or immunoperoxidase staining, both methods
 well known in the art. Antibody from positive clones is used to develop
 kit format diagnostic assays.
 Development of immunoassays suitable for diagnostic and research purposes
 from antibody and antigen is well known in the art. Particular
 considerations for immunoassay development beyond the scope of this
 application are detailed in a review by Nakamura et al., 1992.
 Immunochemical Assays and Biosensor Technology for the 1990's. Am. Soc.
 Microbiol., Wash. D.C.
 While particular embodiments of the invention have been described in
 detail, it will be apparent to those skilled in the art that these
 embodiments are exemplary rather than limiting, and the true scope of the
 invention is that defined in the following claims.