Markers for invasive prostatic neoplasia

This invention is directed to the identification, isolation and use of nonprostate derived markers, such as markers derived from the seminal vesicles, and antibodies which recognize these markers in the diagnosis of invasive proatic neoplasia, to diagnostic aids for screening biological samples for evidence of invasive prostatic neoplasia, and to methods for the use of these diagnostic aids.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to the identification, isolation and use of markers 
and antibodies which recognize these markers in the diagnosis of invasive 
prostatic neoplasia in humans, and to diagnostic aids for screening 
biological samples for evidence of invasive prostatic neoplasia. 
2. Description of the Background 
The prostate, an organ of the mammalian male urogenital system, is located 
at the base of the bladder surrounding the urethra. Although encapsulated, 
the walnut-sized prostate can be divided into five lobes, the posterior, 
middle, and exterior lobes and two lateral lobes. Histological examination 
reveals that the prostate is a highly microvascularized gland comprising 
fairly large glandular spaces lined with epithelium. The majority of fluid 
of the male ejaculate is supplied by this gland and the seminal vesicles. 
The prostate is an endocrine-dependent organ which responds to both the 
major male hormone, testosterone, and the major female hormones, estrogen 
and progesterone. In particular, the testicular androgen is believed 
important for prostate growth and development because, in both humans and 
other animals, castration leads to prostate atrophy and an absence of any 
incidence of prostatic carcinoma. 
There are two major neoplasia of the prostate, benign enlargement of the 
prostate or nodular hyperplasia (also called benign prostatic hyperplasia 
(BPH) or benign prostatic hypertrophy), and prostatic carcinoma. Nodular 
hyperplasia is very common in men over the age of 50. It is characterized 
by the presence of a number of large distinct nodules in the periurethral 
area of the prostate. Although benign, these nodules can produce 
obstruction of the urethra causing nocturia, micturition, and difficulty 
in starting and stopping a urine stream upon voiding the bladder. 
Occasionally, catheterization is required and even surgery. In the more 
extreme cases, secondary changes in the bladder can occur such as 
hypertrophy, acute retention with secondary urinary tract involvement, 
azotemia and uremia. Although all of these changes of the prostate may 
suggest pre-malignancy, there is as yet no direct association between 
nodular hyperplasia and prostatic carcinoma. 
Carcinoma of the prostate is the most common form of cancer in human males 
with upwards of one third of those cases being fatal. In the more 
aggressive forms, transformed prostatic tissue escapes from the prostate 
capsule invading locally and throughout the bloodstream. Local invasions 
typically involve the seminal vesicles, the base of the urinary bladder, 
and the urethra. Hematogenous spread occurs primarily to the bones and 
lymph nodes, but can include massive visceral invasion as well. 
Histologically, most lesions are adenocarcinomas with well-defined gland 
patterns, but the more typical malignancy patterns associated with the 
very aggressive cancers are also common. Except in rare instances, all 
forms of prostatic carcinoma originate in the peripheral zone of the gland 
which is palpable upon rectal examination. 
Prostatic carcinomas are graded and staged by number and letter according 
to histological criteria, the arrangement and appearance of malignant 
glands, and the degree of anaplasia of the cancerous cells. Stage A1 
tumors include the incidental or clinically unsuspected cancers. These are 
detected in autopsy and rarely pose a problem to the patient. Stage B 
tumors are detectable by rectal digital examination and are also confined 
to the prostate. Tumors classified as B1, B2, and so on, indicate 
increasing severity of tumor formation. These tumors are fairly common in 
older men who begin to show signs and symptoms characteristic of some form 
of prostatic carcinoma. Stage C tumors have breached the prostate capsule 
and may or may not have invaded the surrounding tissues such as the 
seminal vesicles. Those tumors which have seminal vesicle involvement show 
an 80% correlation with lymph node involvement (C2). Stage D tumors have 
distinct metastases and a 100% correlation with lymph node involvement. 
Over 75% of patients with prostatic carcinoma show signs of stage C or D 
type development with significant urinary tract involvement. Only 5-10% of 
stage A patients, of those who have been followed for 8-10 years, develop 
stage C or D type prostatic carcinoma although the probability increases 
for patients who first :present at a fairly young age. Young males with 
nodular hyperplasia are typically recommended for surgery or more 
aggressive endocrine therapy. 
Little is known about the causes of prostatic carcinoma, but there are at 
least three confirmed risk factors--age, race and endocrine system. As 
discussed, the incidence of all forms of prostatic neoplasia is very high 
in men over 50. In the 45-49 year old age group the incidence is about 4.8 
per one hundred thousand men and increases to 513 between the ages of 70 
to 75. The incidence of latent carcinoma is higher still. Over 30% of 
prostate tissue in autopsied males over 50 shows some sign of latent 
carcinoma. 
The second risk factor, race, is fairly strong. Among white males in the 
United States the incidence of prostatic neoplasia in those over 50 is 
about 58 per one hundred thousand men. The rate increases to about 95 per 
one hundred thousand in black males whereas in oriental males, prostatic 
neoplasia is rather rare at about 3 to 4 per one hundred thousand in one 
study performed in Hong Kong. The exact reason for this distribution is 
unclear. Although environmental effects should not be discounted, 
epidemiology points to a strong genetic influence. 
The final risk factor, the endocrine system, may be the most important. 
Although, no direct link has been established between absolute or relative 
levels of any hormone and neoplasia of the prostate, the evidence for some 
form of hormonal regulation is convincing. First, in both humans and dogs, 
the only other mammal known to develop hyperplasia with aging, nodular 
hyperplasia or full-blown carcinoma of the prostate only develop in the 
presence of intact testes. Secondly, in castrated young dogs it is 
possible to induce nodular hyperplasia by administering of androgen and 
estradiol, suggesting that hormones produced by the testes are required 
for prostate development. Further, there is some evidence that 
dihydrotestosterone, which is derived from testosterone, may be the 
ultimate mediator of cell growth. Prostate cells of the epithelium are 
covered with dihydrotestosterone receptors which increase in number in the 
presence of estrogen. In men and dogs, plasma testosterone levels decrease 
and estradiol levels increase with age. This alteration shifts the 
hormonal balance of the cells and possibly sensitizes the prostate for 
transformation. At the very least, it appears that androgens are required 
to maintain the viability of prostate epithelium from which most 
carcinomas derive. 
Yearly rectal examination is very useful for the early detection of 
prostatic neoplasia. This detection method is fairly simple and 
straightforward. However, it is subject to bias and not very well 
standardized. At the earliest, it can only detect stage B carcinoma and 
has no capacity to determine whether stages C or D are developing. 
Further, the digital rectal exam is not very sensitive. Approximately 
30-60% of men have a prostatic neoplasia that cannot be detected by the 
physician, which is further complicated by the fact that these men usually 
present with no symptoms at all. A number of new techniques look 
promising. These include ultrasound and other methods of noninvasive 
detection such as positron emission tomography (PET). These methods are 
limited to the detection of formed tumors and are unable to detect 
prostatic carcinoma which is just beginning to invade surrounding tissue. 
Chemotherapy, surgery or radiotherapy is the treatment of choice for stage 
A or B prostatic neoplasia. Surgery involves complete removal of the 
entire prostate, radical prostatectomy, and often removal of the 
surrounding lymph nodes, lymphadenectomy. Radiotherapy may be either 
external or interstitial using .sup.125 I and is typically performed in 
conjunction with surgery. Endocrine therapy is the treatment of choice for 
more advanced forms. The aim of this therapy is to deprive the prostate 
cells, and presumably the transformed prostate cells as well, of 
testosterone. This is accomplished by administering estrogens or synthetic 
hormones which are agonists of luteinizing hormone-releasing hormone 
(LHRH). These cellular messengers directly inhibit testicular and organ 
synthesis and suppress luteinizing hormone (LH) secretion which in turn 
leads to reduced testosterone secretion by the testes. Despite the 
advances made in achieving a pharmacologic orchiectomy, the survival rates 
for those with stage C and D carcinomas are rather bleak. In the short 
term, the most promising results will be achieved by earlier detection 
using more sensitive assays. 
Carcinoma cell invasion of the seminal vesicles is a very poor prognosis 
for the patient. As discussed, seminal vesicle involvement frequently 
correlates with metastases to the lymph nodes and subsequent dissemination 
throughout the body. Invasion of the seminal vesicles begins with cell 
multiplication at the base of the prostate. Transformed cells expand 
within and through the ejaculating duct, localizing in the seminal 
vesicles near their point of junction with the vas deferens (A. A. Villers 
et al., J. Urol. 143:1183, 1990). Surprisingly, others have found a 
relatively low frequency of positive nodes in patients with seminal 
vesicle invasion, but a comparable prognosis among patients with and 
without lymph node metastases (E. Mukamel et al., Cancer 59: 1535, 1987). 
No alternative explanation was proposed. Uncertainty in these results may 
stem from the fact that the seminal vesicles are not very well defined 
morphologically or biochemically. 
There are two seminal vesicles in man, one located on each side of the 
urethra posterior to the urinary bladder and superior to the prostate. 
They are believed to contain two types of epithelial cells, principal or 
superficial cells, and basal cells. Each gland is open to the urethra and 
comprises a highly convoluted tube coiled upon itself which, if extended, 
would be approximately 15 cm in length. The convolutions within each gland 
impart a honeycomb appearance when viewed under cross section. The 
internal cells of the individual vesicles are highly interconnected with 
ridges and folds both circular and longitudinal. Individual cells of the 
walls contain numerous secretory bodies including golgi vacuoles, electron 
dense granules and droplets. These bodies secrete a mixture of materials 
into the lumen of each tubule. Approximately 70% of human ejaculate is 
composed of this material which contains fructose, citrate, inositol, 
prostaglandin, choline esters, and a number of soluble proteins. A few of 
these proteins have been identified as specific to seminal vesicle tissue 
including semenogelin I, a large molecular weight protein which can be 
broken down into three subunits of 52 kDa, 71 kDa, and 76 kDa (H. Lilja et 
al., J. Biol. Chem. 264:1894, 1989), semenogelin II (H. Lilja and A. 
Lundwall, Proc. Natl. Acad. Sci. USA 89:4559, 1992), lactoferrin or 
scafferin (A. Hekman and P. Rumke, Fertil. Steril. 20: 312,1969), seminal 
vesicle specific antigen (SVS A), MHS-5 specific antigen (J. C. Herr et 
al., J. Reprod. Immunol. 16:99, 1989), rat seminal vesicle specific (SVS) 
proteins I-VIII (J. Seitz and G. Aumuller, Andrologia 22:25, 1990), 
B-microseminoprotein (B-MSP) (K. Akiyama et al., Biochim. Biophys. Acta 
829:288, 1985), and seminal plasma number 7 antigen (K. Koyama et al., J. 
Reprod. Immunol. 5:134, 1983). These proteins and antigens are only now 
being analyzed in detail and some have been cloned by recombinant DNA 
techniques. 
Fairly recently, a number of serum antigens have been characterized as 
markers for prostatic neoplasia. These markers are useful because they are 
relatively straightforward to assay using noninvasive procedures and may 
detect prostatic neoplasia at very early stages of development. Both 
malignant and normal prostate epithelial cells were found to express a 
prostate-specific acid phosphatase (PAP) which is detectable in serum by 
biochemical and other immunological techniques. Elevated PAP levels 
correlate well with neoplasia that has spread beyond the prostate capsule. 
Consequently, PAP is a useful serum marker for characterizing the later 
stages of prostatic neoplasia and also for monitoring the progress of the 
disease in patients. 
Another marker which has proved to be of value is the prostate-specific 
antigen (PSA), a serine protease found in both normal and neoplastic 
prostate epithelium. Investigations have determined that there is a direct 
correlation between serum PSA levels with the size and stage of a tumor. 
The normal concentration of PSA in men is from 0 to 2.8 ng/ml of serum. In 
one study, researchers determined that average PSA concentrations in the 
serum of patients grouped according to severity were proportional to the 
clinical state of the tumor (T. A. Stamey, et al., N. Engl. J. Med. 
317:909, 1987). These authors did not indicate whether PSA levels could be 
used to determine the pathological stage of carcinoma in individual 
patients. Concentrations of 40 ng/ml were predictive of advanced stages of 
disease, but the predictive value of serum concentrations of less then 15 
ng/ml were less than clear. PSA titers were only marginally useful to 
distinguish whether the tumor was contained by or had escaped the 
prostate. Levels greater than 10 ng/ml were typical in patient groups with 
more advanced and gland-unconfined carcinomas. However, it was not 
atypical to find high PSA levels in patient groups with gland-confined 
hyperplasia. 
These theories were partly confirmed in a more recent study which looked at 
serum PSA levels in 209 men with various stages of prostatic neoplasia (T. 
E. Osterling et al., J. Urol. 139:766, 1988). These authors determined 
that PSA levels showed a statistically significant correlation with 
pathological stages when compared within the various groups. However, the 
levels were far less useful when looking at patients on an individual 
basis. There was a large degree of variability between patient groups and 
a significant number of both false and missed positives. In a rigorous 
analysis using greater numbers of men and taking into account actual or 
predicted numbers of carcinoma cells, Partin et al. determined that serum 
PSA levels were influenced by tumor volume and the stage of 
differentiation (A. W. Partin et al., J. Urol. 143:747, 1990). Mean 
antigen levels increased with advanced pathological stage, but this seemed 
to be related more to overall tumor volume than to any particular stage of 
the disease. In fact, immunohistochemical studies revealed that higher 
stage tumors actually produced less PSA, possibly due to the diseased 
state of the cells. The authors concluded that PSA levels are unreliable 
for preoperative prediction of the pathological stage of individual 
patients. 
A number of other prostate antigens have since been identified. The most 
well-studied of these has been the prostatic carcinoma associated complex 
() also called the glycoprotein complex (G. L. Wright et al., Int. J. 
Cancer 47:717, 1991). Although specific for prostatic epithelium, this 
protein complex of 35-310 kDa antigens was not correlative for the staging 
of prostatic carcinoma. 
SUMMARY OF THE INVENTION 
The present invention overcomes the problems and disadvantages associated 
with current strategies and designs and provides a new method for 
stagespecific detection of prostatic neoplasia in a patient. 
As broadly described herein, one embodiment of the invention is directed to 
a method for identifying, isolating, and using markers derived from 
non-prostatic tissues such as the seminal vesicles in the detection of 
prostatic neoplasia in a patient. The marker may comprise a protein or an 
antigenic part of a protein. This invention also encompasses the 
identification, isolation and cloning of the gene or genes which code for 
the specific marker. The recombinant gene or genetic sequence is used to 
express recombinant marker or antigenic parts of the marker. 
As broadly described herein, another embodiment of the invention is 
directed to methods for the identification and isolation of antibodies to 
seminal vesicle-specific markers and other non-prostatic markers in 
biological samples. Marker is incubated with a biological sample taken 
from a patient suspected of having prostatic neoplasia. The sample may be 
tissue, blood, urine, or semen. The amount of serum-derived antibody which 
binds to the marker is determined and, if over a predetermined base level, 
indicates the presence of specific antibody in the sample and prostatic 
neoplasia in the patient. 
As broadly described herein, a further embodiment of the invention is 
directed to an antibody which specifically binds to the marker and to a 
method for using this antibody to detect prostatic neoplasia in a patient. 
The antibody may be a monoclonal or polyclonal antibody or a fragment of a 
monoclonal or polyclonal antibody such as an Fv fragment and preferably 
the antibody is an IgG isotype. In one aspect of the invention the 
specific antibody is incubated with a biological sample taken from the 
patient suspected of having prostatic neoplasia. The sample may be tissue, 
blood, urine, or semen. The amount of specific marker in the sample which 
binds to the antibody is determined and if over a predetermined base level 
indicates the presence of specific marker in the sample and prostatic 
neoplasia in the patient. 
As broadly described herein, a still further embodiment of the invention is 
directed to diagnostic kits for the detection of prostatic neoplasia in a 
patient comprising the marker or the marker-specific antibody and methods 
for using these markers and antibodies for the detection of prostatic 
neoplasia. 
Other objects and advantages of the invention are set forth in part in the 
description which follows, and in part, will be obvious from this 
description, or may be learned from the practice of the invention.

DESCRIPTION OF THE INVENTION 
To achieve the objects and in accordance with the purposes of the 
invention, as embodied and broadly described herein, the present invention 
comprises markers, parts of markers, genes and genetic sequences which 
encode these markers, both monoclonal and polyclonal antibodies and parts 
of antibodies, and diagnostic kits for the detection of prostatic 
neoplasia in a patient. 
Neoplasia of the prostate can be divided into two basic forms, nodular 
hyperplasia and carcinoma. Nodular hyperplasia is not a serious health 
concern. For those with asymptomatic hyperplasia, no treatment is 
necessary. For those with symptomatic hyperplasia of the prostate, therapy 
typically involves chemotherapeutic drugs, radiation therapy, or radical 
prostatectomy. Prostatic neoplasia only becomes life threatening when 
transformed prostate cells break through the prostate capsule and 
metastasize throughout the body. Therefore, it is the invasion-proficient 
status which is most important in the detection of this disease. 
The seminal vesicles are often invaded by prostatic carcinoma cells. Upon 
invasion this normally highly organized and compartmentalized structure 
becomes damaged. Damage due to physical disruption of the seminal vesicles 
results in the presentation of novel seminal vesicle-derived markers to 
the blood stream and other bodily fluids. In one aspect of the invention, 
these disrupted and damaged cells passively release seminal 
vesicle-specific macromolecules, or markers, which may be proteins, 
cytokines, modified proteins, peptides, complex biochemicals, fragments of 
proteins or peptides, nucleic acids, or modifications or combinations 
thereof, to areas of the body such as, for example, the bloodstream. In 
another aspect, the damaged and "leaky" vasculature produced by the 
invading carcinoma leads to the direct release of seminal vesicle 
secretions into areas of the body such as, for example, the semen, urine 
or bloodstream. In either situation, once released, these markers may be 
detectable by biochemical techniques known to those of ordinary skill in 
the art. 
These seminal vesicle-derived markers are also likely to be highly 
antigenic. The patient's lymphocytes will produce antibodies specific to 
these "newly-recognized" seminal vesicle-derived antigens. Although the 
seminal vesicles are not foreign to the patient, antigens released have 
not previously been exposed to the host's immune system and could 
stimulate a humoral or cellular response. These host-derived, 
antigen-specific antibodies and/or antigen-primed cells may also be 
detectable by biochemical techniques known to those of ordinary skill in 
the art. Upon detection and quantitation, the absolute or relative amounts 
of seminal vesicle-specific antigens or antibodies may be determinative of 
a particular stage of prostatic neoplasia. These results may be used alone 
or in combination with PAP and/or PSA titers to select or rule out a 
course of therapy for a patient. 
A first embodiment of the invention is directed to the identification of 
seminal vesicle-specific markers, which may be proteins, cytokines, 
modified proteins, peptides, complex biochemicals, fragments of proteins 
or peptides, nucleic acids, or modifications or combinations thereof, and 
are useful for the detection of prostatic neoplasia. First, seminal 
vesicle-specific markers are identified and isolated. For example, seminal 
vesicles or tissue samples containing seminal vesicle-specific markers 
such as blood, semen, or urine are obtained. In a direct approach, seminal 
vesicle tissue is isolated by necropsy from, for example, human cadavers 
or by radical prostatectomy. Samples may be used immediately or frozen to 
-80.degree. C. for later use. 
Samples are fractionated by, for example, chromatography, such as 
ion-exchange or affinity column chromatography, salt fractionation using 
for example ammonium sulfate precipitation, centrifugation, size and 
chemical fractionation, SDS-polyacrylamide gel electrophoresis (PAGE) run 
under reducing or non-reducing conditions, or filtration. Using such 
techniques, particular fractions or extracts containing, for example, the 
glycoprotein-rich secretory markers, can be targeted. Alternatively, or in 
addition to these procedures, more rigorous techniques can be used to 
isolate single markers or antigens or groups of markers, such as 
high-performance liquid chromatography (HPLC), reversed-phase HPLC, ion 
exchange HPLC, fast-phase liquid chromatography (FPLC), one-, two-, or 
three-dimensional electrophoresis followed by electro-elution or 
electrotransfer of the markers of interest from the electrophoresis matrix 
onto a membrane such as a nitrocellulose membrane. These and other 
so-called conventional techniques for the isolation of proteins and 
peptides are described in Proteins: Structures and Molecular Properties 
(T. E. Creighton, Freeman and Co., N.Y., 1984), and A Practical Guide to 
Protein and Peptide Purification for Micro Sequencing (P. T. Matsudaira, 
Academic Press, N.Y., 1989), which are hereby specifically incorporated by 
reference. 
In an indirect-approach, seminal vesicle tissue can be used to create 
antibodies specific to seminal vesicle markers which are used to identify 
and isolate those antigenic markers. For example, seminal vesicle tissue 
or biological samples from patients with suspected or confirmed cases of 
some form of prostatic neoplasia are treated to isolate fractions which 
are likely to contain seminal vesicle-specific markers. These include, for 
example, fractions of cell surface antigens, glycoproteins, and 
lipoproteins, or fractions of cells disrupted to release 
membrane-associated and cytoplasmic antigens. Each of these fractions is 
injected into a female laboratory animal, such as a rabbit, a guinea pig, 
a rat or a mouse, to create seminal vesicle-specific polyclonal or 
monoclonal antibodies. Female animals are chosen as these are believed to 
have the lowest probability of containing anti-seminal vesicle antibodies 
and the highest probability of generating a strong immune response. After 
injection and possibly the administration of booster injections, blood is 
collected and polyclonal antisera and/or antibodies are isolated from the 
serum. If necessary, seminal vesicle specific antibodies can be purified 
using, for example, affinity chromatography. 
Monoclonal antibodies are also prepared. About three to four weeks after 
the initial injections, spleen cells are isolated, fused with myeloma 
cells of the same or a different species, such as for example, the murine 
cell lines P3-X63 Ag8, X63Ag.653, SP2/0-Ag14, FO, NSI/1-Ag4-1, NSO/1, and 
FOX-NY, or the rat cell lines Y3-Ag.1.2.3, YB2/0, and IR983F, and screened 
for hybridomas which express seminal vesicle-specific monoclonal 
antibodies. Hybridomas expressing human antibody or mostly human antibody 
can be creating by fusing the spleen cells obtained with human myeloma or 
human heteromyeloma cells such as, for example, U-266, FU-266, and HFB-1. 
A fusion procedure which employs polyethylene glycol or Epstein-Barr virus 
is preferred. Methods for the creation of antigen-specific polyclonal and 
monoclonal antibodies are disclosed in Antibodies: A Laboratory Manual (E. 
Harlow and D. Lane, Cold Spring Harbor, 1988). These antibodies are used 
to detect markers which are specific to seminal vesicles by 
immunoprecipitation, immunoblotting, such as Western blotting of 
electrophoresed seminal vesicle-specific antigens, or affinity 
chromatography. 
Alternatively, seminal vesicle-specific markers, which may be purified, 
partially purified, or recombinantly produced, can be used to identify and 
isolate seminal vesicle-specific antibodies in, for example, the blood 
stream of patients. Seminal vesicle-specific markers are coupled to a 
matrix such as, for example, sepharose, sephadex, sephacel, or sephacryl, 
using techniques which are known to those of ordinary skill in the art. 
Whole blood or preferably blood plasma is subjected to, for example, 
affinity column chromatography using the antigert-coupled matrix. 
Fractions comprising seminal vesicle-specific antibodies are eluted and 
further purified using affinity chromatography with a purified 
antigen-coupled matrix or using conventional techniques such as 
differential centrifugation or other known separation techniques. 
Seminal vesicle-specific markers which are identified and isolated, at 
least partially, are characterized. Their molecular weight is determined 
by, for example SDS-PAGE. The isoelectric point is determined by 
2-dimensional PAGE techniques such as isoelectric focusing. The amino acid 
sequence is determined by, for example, partial digestion of the purified 
antigen, if necessary, automated sequence analysis of the digestion 
products, and reconstruction of the complete sequence from the resulting 
data. The presence or absence of lipids, carbohydrates, unusual amino acid 
residues, polysaccharide and other modifications is also determined. From 
knowledge of the complete sequence of a macromolecule, such as a protein 
or peptide, hydrophobicity-hydrophilicity charts can be determined, 
particularly antigenic regions identified, and three-dimensional 
structures predicted. Once characterized, these markers are compared with 
other known seminal vesicle-specific molecules either by computer sequence 
alignment analysis or by direct comparison of know features and, if 
necessary, side-by-side characterization. 
With knowledge of even a portion of the amino acid sequence of the marker, 
it is possible to determine the genetic sequence with codes for the entire 
marker and to clone this sequence from the cellular genome or chemically 
synthesize all or part of the gene. To clone the gene, a genomic or cDNA 
library is created and probed with the genetic sequence of interest, which 
may be chemically synthesized or isolated from a genetic library. Positive 
clones are picked, expanded, and expressed to identify their products. In 
this fashion, an entire gene can be cloned from a cellular genome and 
expressed in a recombinant expression vector. Alternatively, using 
polyclonal or monoclonal antibodies, cDNA expression libraries can be 
probed for seminal vesicle-specific markers directly. The positive clones 
are identified, expanded, and their recombinant DNA sequences subcloned 
using techniques which are well known for those of orclinary skill in the 
art such as, for example, those described in Current Protocols in 
Molecular Biology (F. M. Ausubel et al., Green Publishing Assoc. and 
Wiley-Interscience, 1989), and Molecular Cloning: A Laboratory Manual, 
2.sup.nd Ed. (J. Sambrook et al., Cold Spring Harbor Laboratory, N.Y., 
1989), which are hereby specifically incorporated by reference. 
Recombinant vectors containing all or specific antigenic portions of the 
gene of interest are created and used to produce large quantities of 
recombinant expression product in prokaryotes, such as, for example, E. 
coli, eukaryotes such as, for example, Bacculovirus, plant cells, or 
animal cells, or yeast cells. The amino acid sequence of the gene of 
interest is also synthesized chemically to produce large quantities of 
marker and to isolate additional marker. 
Markers, including antibodies, antigens, and antibody or antigen fragments 
produced accordingly are useful in diagnostic kits for the detection of 
invasive prostatic neoplasia. As discussed, invasive prostatic neoplasia 
may be associated with release of seminal vesicle-specific antigens into 
areas of the body which do not normally receive these antigens, such as, 
for example, the bloodstream, the bladder, or the passageways of the male 
urogenital system such as the vas deferens, the bulbourethral gland, or 
the urethra. A sample of biological fluid, such as, for example, a tissue 
sample, or a sample of biological fluid such as semen, urine or blood, 
taken from the patient suspected of having prostatic neoplasia is analyzed 
for the presence or the increased presence of one or more of these seminal 
vesicle-specific markers or antigens or for the presence or increased 
presence of human antibodies directed against such markers or antigens. 
Another embodiment of the invention is the analysis of samples of tissue or 
biological fluid obtained from patients suspected of having prostatic 
neoplasia for the presence of seminal vesicle-specific antigens as markers 
for invasive prostatic neoplasia. Useful assays include, for example, an 
enzyme immune assay (EIA) such as an enzyme-linked immunosorbent assay 
(ELISA), a radioimmune assay (RIA), a Western blot assay, or a slot blot 
assay. Briefly, samples of biological fluid believed to contain one or 
more markers of invasive prostatic neoplasia are incubated with seminal 
vesicle-specific antibody prepared according to the invention. The 
antibody may be of any isotype including IgG.sub.1, IgG.sub.2a, 
IgG.sub.2b, IgM, IgA, or IgD, but an IgG is preferable. Optionally, the 
antibody, which may be polyclonal, monoclonal, or a fragment of a 
monoclonal or polyclonal antibody, preferably the Fv fragment, may be 
fixed to a solid support to facilitate washing and subsequent isolation of 
the complex. Examples of solid supports include glass or plastic such as, 
for example, a tissue culture plate, a vial, a microtiter plate, a stick, 
a paddle, a bead, or a microbead. After incubation, the mixture is washed 
and the amount of antibody-antigen complex formed determined. This is 
accomplished by incubating the washed mixture with a second, labeled 
antibody which is specific to the complex. This second antibody may be a 
monoclonal or polyclonal antibody and is labeled with a detectable label. 
Examples of detectable labels include a radio isotope, a stable isotope, a 
fluorescent chemical, a luminescent chemical, a metal, an electrical 
charge, an enzyme, a chromatic chemical, a spatial chemical, an 
electron-dense molecule, or a label detectable by mass spectrometry. 
Alternatively, the amount of seminal vesicle-specific antigert in the 
sample may be determined using an indirect assay, wherein, for example, a 
second, labeled antibody is used to detect bound seminal vesicle-specific 
antibody, and/or in a competition or inhibition assay wherein, for 
example, a monoclonal antibody which binds to a distinct epitope of the 
antigen are incubated simultaneously with the mixture. Each of these 
assays are well known to those of ordinary skill in the art and described 
in, for example, Antibodies: A Laboratory Manual. 
In another embodiment of the invention, a biological sample is taken from a 
patient suspected of having prostatic neoplasia and assayed for the 
presence of host antibodies to seminal vesicle-specific antigens. Useful 
assays include, for example, an RIA, an EIA such as an ELISA, a Western 
blot or a slot blot. As discussed, seminal vesicle antigens may be 
released into the body or blood stream upon invasion of transformed 
prostatic cells. These antigens may be recognized as foreign by the immune 
system of the body and anti-antigen antibodies produced. These host 
antibodies can be specifically detected using a diagnostic kit which 
comprises one or more purified or partially purified seminal 
vesicle-specific markers. As before, the assay to detect these antibodies 
can be a direct, indirect, competitive or inhibition assay. The assay may 
comprise polyclonal antibodies or fragments of polyclonal antibodies, such 
as the Fv fragment, or monoclonal antibodies or fragments of monoclonal 
antibodies, such as the Fv fragment, either of which are labeled with a 
detectable label such as, for example a radio isotope, a stable isotope, a 
fluorescent chemical, a luminescent chemical, a metal, an electrical 
charge, an enzyme, a chromatic chemical, a spatial chemical, an 
electrondense molecule, or a label detectable by mass spectrometry. The 
antibodies to be detected may be of any isotype including IgG, IgG.sub.2a, 
IgG.sub.2b, IgM, IgD, IgA, or a combination of these isotypes. Isotype 
specific anti-antibodies may also be utilized to identify or quantitate 
specific antibody isotypes. 
In a direct assay, it is preferable that the seminal vesicle-specific 
antigens or antigenie fragments be produced recombinantly, although as 
discussed, antigens can also be produced synthetically or isolated and 
purified by convention techniques. It is preferred that the marker, or 
antigen, be fixed for a solid support such as, for example, glass or 
plastic such as a tissue culture plate, a vial, a microtiter plate, a 
stick, a paddle, a bead, or a microbead. A biological sample suspected of 
containing marker-specific antibody is added to the fixed antigen or 
marker and incubated, for example, between one hour and overnight: in 
phosphate-buffered saline (PBS), at between 4.degree. C. and 37.degree. 
C., preferably at about room temperature. After incubation, the mixture is 
removed and the solid support washed. The washed support is incubated 
with, for example, a labeled anti-antibody and incubated as before. The 
label is detectable and may be a radio isotope, a stable isotope, a 
fluorescent chemical, a luminescent chemical, a metal, an electrical 
charge, an enzyme, a chromatic chemical, a spatial chemical, an 
electron-dense molecule, or a label detectable by mass spectrometry. The 
amount of labeled antibody which binds to the solid support is determined 
and compared to the amount of labeled, non-specific antibody which remains 
bound in control assays. Control assays comprise assays that test 
biological samples which are known not to contain marker-specific antibody 
or another assay which provides a determination of background levels of 
antibody and/or a baseline internal negative control. If the amount of 
antibody bound is greater than a predetermined background or base level, 
the biological sample contains seminal vesicle-specific antibodies and the 
patient is diagnosed as likely to have invasive prostatic neoplasia. 
Alternatively, the assay can be performed without washing and without 
separate incubation steps by using polyclonal or monoclonal antibodies. 
For example, using a competition assay, excess labeled antibody is added 
to antigens fixed to a solid support. This provides a measure of 100% 
binding. To another sample or series of samples, biological samples, such 
as samples of serum, are added to the incubation mixture and any decrease 
from 100% binding is indicative of the presence of seminal 
vesicle-specific antibodies in the sample. 
This invention also comprises procedures and techniques to identify, 
isolate, and utilize markers derived from tissues other than the seminal 
vesicles for the detection of invasive prostatic neoplasia. Likely tissues 
include those tissues which surround and are in close proximity with the 
prostate such as the prostate capsule, the ejaculatory duct, the bladder, 
the vas deferens, the bulbourethral gland, the crus, the urethra, the 
corpus spongiosum, and the corpus cavernosum. Markers may be passively 
released upon tissue damage by the invading prostatic cells or actively 
released into new areas of the body. The non-prostate, non-seminal 
vesicle-derived markers may be proteins, cytokines, modified proteins, 
peptides, complex biochemicals, fragments of proteins or peptides, nucleic 
acids, or modifications or combinations thereof. As before, likely markers 
of prostate neoplasia may be identified in biological samples, such as 
tissue, blood, semen, or urine, and traced back to one or more of these 
tissues. Alternatively, these tissues can be fractionated and screened for 
likely markers and these markers used in diagnostic kits and methods 
utilizing procedures described herein. In addition, host reactions, both 
humoral and cellular, would occur against passively or actively released 
markers. Antibodies to these markers could also be detected in diagnostic 
kits and are also described herein. 
The following examples are offered to illustrate embodiment of the present 
invention, but should not be viewed as limiting the scope of the 
invention. 
EXAMPLES 
Example 1 - Identification of Seminal Vesicle-Specific Markers 
Seminal vesicle tissue is surgically removed from a human cadaver or 
removed by radical prostatectomy biopsy from a patient to a 100 mm tissue 
culture dish containing about 10 ml of cold (4.degree. C.) hypertonic 
buffer (50 mM Tris-Cl, pH 7.0; 1.0 mM KCl; 1 mM PMSF; 10 mM EDTA; +/-1 mM 
DTT). Tissue is mechanically minced and/or dispersed by passage through a 
19 gauge needle, followed by homogenization using either a cylindrical 
homogenizer or rotary tissue disrupter. The membrane fractions and soluble 
fractions of samples are separated by centrifugation. The pelleted 
fraction is suspended in hypotonic buffer plus 2% SDS and centrifugation 
is repeated. This process is diagrammatically outlined in FIG. 1. 
Optionally, lectin affinity chromatography may be used to enrich the 
soluble fractions for glycoproteins (as predictive for secretory 
proteins). 
Semen, urine, and blood samples are collected from normal volunteers and 
patients with suspected or confirmed cases of prostatic carcinoma at 
various stages of severity. Semen ejaculate samples, preferably from 
normal vasectomized individuals, are mixed immediately with PMSF and EDTA 
in chilled phosphate buffered saline, pH 7.2 (PBS) or hypotonic buffer. 
These samples are separated into soluble and particulate fractions by 
pelleting at 10,000.times.g for 10 minutes. The particulate fraction may 
be suspended in hypotonic buffer and diluted as necessary to about 1 
ug/100 ul in PBS. The soluble fraction may be concentrated using Amicon 
filters or diluted with PBS as necessary to about 1 ug/100 ul of liquid. 
Seminal vesicle fractions including the membrane fraction, the soluble 
fraction and the enriched fraction, and fractions of blood and semen are 
subjected to SDS polyacrylamide gel electrophoresis (PAGE) under reducing 
or non-reducing conditions, along with appropriate molecular weight 
markers and negative controls. SDS-PAGE gels have an extended longitudinal 
dimension (ca. 16") for enhanced band resolution. The resulting gel 
matrices are subjected to electrotransfer to a suitable membrance such as 
nitrocellulose. 
Briefly, the electrophoresis matrix is equilibrated for about 30 minutes in 
transfer buffer (18.2 g Tris Base; 86.5 g glycine; 4.0 liters H.sub.2 O; 
1200 ml methanol) at room temperature. Pre-wetted nitrocellulose transfer 
membrane is cut to size and placed on top of the gel. Pre-wetted Whatmann 
3 MM filter papers are placed on both sides of the gel-nitrocellulose 
slab, and the entire structure placed in an electrophoresis tank of 
transfer buffer. Electrophoresis is begun for 30 minutes at 100 volts, 
turned down to 14 volts (constant voltage) and continued overnight (about 
14 hours) at 4.degree. C. Upon completion of the transfer, the membrane is 
removed and assayed for transfer efficiency. 
To visualize transferred proteins, membranes are placed in Ponceau S 
solution (0.5 g Ponceau S dissolved in 1 ml glacial acetic acid and 
brought to a total volume of 100 ml with H.sub.2 O just before use) for 5 
minutes at room temperature. The membranes are destained for 2 minutes in 
water and photographed using transmitted light through the stained 
nitrocellulose membrane. A photograph of a representative stained membrane 
of a high-resolution, 16" PAGE gel is shown in FIG. 2A. Bands observed on 
the membrane are isolated directly or the seminal vesicle-specific 
antigens further identified by probing the membranes with seminal 
vesicle-specific antibody created as follows. Briefly, fractions of 
seminal vesicle tissue, blood and/or semen, created above, are mixed with 
equal volumes of incomplete Freund's adjuvant and injected subcutaneously 
into female rabbits using a 25-gauge needle at a dose of about 400 ul per 
injection. The first antigen only is presented in complete Freund's 
adjuvant. Each animal receives from one to ten injections at various sites 
high on the dorsal flank between the ribs and the hip. Antigen injections 
are repeated approximately every two weeks for a minimum of three times 
using fresh seminal vesicle tissue materials. 
After about four weeks from the initial injection, 5-10 ml samples of blood 
are collected by venipuncture from the rabbits' marginal ear vein using a 
23-gauge needle. Blood flow is stopped by applying gentle pressure to the 
cut with sterile gauze for 10-20 seconds. Collected blood is allowed to 
clot for a minimum of one hour at room temperature. The clotted material 
is removed with sterile tweezers and any remaining solid material removed 
by centrifugation at 10,000.times.g for 10 minutes. Serum is stored in 500 
ul aliquots at -80.degree. C. until use. 
Ponceau S stained membranes are completely destained by continuing to soak 
in water for an additional 10 minutes and placed in heat-sealable plastic 
bags or trays with 5 ml of blocking buffer (0.1% Tween 20 in 100 mM 
Tris-Cl, pH 7.5; 0.9% NaCl), for about 30 minutes at room temperature on 
an orbital shaker. Serum samples (including normal pre-immune rabbit serum 
control samples) are thawed, diluted at 1:100 to 1:10,000 in blocking 
buffer, and 5 ml added to the bags or trays along with fresh blocking 
buffer. The membranes are incubated for 30 minutes to overnight at room 
temperature in an orbital shaker. After incubation, the membranes are 
removed and washed in blocking buffer several times for about 15 minutes 
each with agitation. Commercially available alkaline-phosphatase 
conjugated anti-rabbit secondary antibody with fresh blocking buffer is 
added to the bags or trays which are incubated for one hour at room 
temperature in an orbital shaker. After incubation, the buffer is removed 
and the membranes treated according to the appropriate enzymatic 
visualization protocol. 
Bands observed using seminal vesicle specific anti-serum in the blood, 
urine, or semen sample that are also observed in the seminal vesicle 
sample, but not in the negative control samples or using normal rabbit 
serum are considered positive and likely candidates for use in the 
detection of prostatic neoplasia. 
Example 2 - Isolation of Seminal Vesicle-Specific Markers 
Positive bands visualized by Ponceau S staining in Example 1 are directly 
excised from the membrane (FIG. 2B). Excised bands are ground into a 
powder or dissolved in DMSO using the method of Kundson (K. A. Kundson, 
Proteins transferred to nitrocellulose for use as immunogen, 
Annual-Became. 147:285, 1985), mixed with incomplete Freund's adjuvant and 
injected into rabbits as described in Example 1 to generate specific 
antisera. The first antigen only is presented in complete Freund's 
adjuvant. 
This antisera can be used directly to identify seminal vesicle-specific 
markers in patient samples, such as samples of blood, urine, or semen and 
are analyzed by Western blot or can be used to create immunoaffinity 
columns. Briefly, antisera is diluted to about 20 ug/ul in PBS. Two mils 
of serum are dialyzed against one liter of dialysis solution (100 rnM 
NaHCO.sub.3 ; 400 mM NaCl) at 4.degree. C. for 24 hours with three 
solution changes. The dialyzed serum is centrifuged at 100,000.times.g for 
one hour to remove aggregates, the resulting supernatant is diluted to 5 
mg/ml with dialysis solution, and the clarified serum stored at 4.degree. 
C. Commercially available cyanogen bromide (CNBr) activated Sepharose is 
prepared according to the appropriate protocol and coupled with the 
antibodies of the serum. The percent coupling is determined and the 
antibody-coupled Sepharose stored at 4.degree. C. in TSA buffer (10 mM 
Tris-C1, pH 8.0; 140 mM NaCl; 0.025% NAN.sub.3) until use. 
Appropriate fractions of seminal vesicle tissue or fluid, blood, or semen 
are prepared as before and added to columns of antiserum coupled 
Sepharose. The columns are washed with TSA buffer and eluted with 50 mM 
Tris-C1, pH 6.8. Eluted samples are confirmed to be seminal 
vesicle-specific using the procedure described in Example 1. Fractions 
collected from the immunoaffinity columns are subjected to further 
purification using HPLC or reverse-phase HPLC. 
Example 3 - Peptide Sequencing 
Proteins isolated by band excision from HPLC analysis as described in 
Example 2 are analyzed for amino acid composition and sequence. 
Band-excised purified proteins or protease digested fractions of proteins 
are applied to a 470A gas phase protein sequenator (Applied Biosystems, 
Inc.) which is connected to an ABI 120 (HPLC) PTH analyzer. Amino acid 
sequences determined are compared to the GENBANK DNA and NBRF protein data 
bases on a Macintosh IIsi personal computer using MacVector version 4.0 
software to determine if the proteins have been previously identified. 
Based on the sequences determined, corresponding peptide sequences are 
prepared. Synthetic peptides are coupled to keyhole lymphocyte hemocyanin 
(KLH) using commercially available kits (Pierce Chem. Co.) to facilitate 
anti-peptide antibody production in rabbits or mice. 
Example 4 - Production of Seminal Vesicle Marker-Specific Monoclonal 
Antibodies 
Purified and synthetic proteins and peptides are individually 
intraperitoneally injected into Balb/c mice in an equal volume of 
incomplete Freund's adjuvant at a total volume of about 250 ul per 
injection. Identical booster injections are given at three-week intervals. 
Three days after the final booster, the mouse is sacrificed and the spleen 
removed and placed in a 100 mm sterile culture dish with about 10 ml of 
RPMI 1640 medium without serum. Spleen cells are teased and torn apart 
using a pair of 19 gauge needles and aspirated until the cells are fully 
dispersed. The cell suspension is allowed to sit for three minutes for 
large clumps to settle and the suspended cells removed. Cells are washed 
twice by centrifugation at 400.times.g in RPMI 1640 at 37.degree. C. in 
the absence of serum. P3-X63Ag8 myeloma cells of equal number are also 
washed twice in serum free medium at 800.times.g. After the final wash, 
the two cell pellets (myeloma and spleen cells) are combined in serum-free 
medium pre-warmed to 37.degree. C. and centrifuged at 800.times.g for 5 
minutes. All medium is carefully removed from the pellet, which is 
suspended in a solution of 50% PEG by slowly adding the PEG while slowly 
stirring the cell pellet with the end of a piper for one minute. Stirring 
is continued for another minute. Pre-warmed serum free medium is added to 
the cell suspension slowly over the next 3 minutes to a total volume of 10 
mils. Cells are centrifuged for 5 minutes at 800.times.g and resuspended 
in 10 mils of medium supplemented with 10% fetal calf serum. Cell 
suspensions of 100 ul each are transferred into wells of a 96-well 
microtiter plate, incubated at 37.degree. C. in a 5% CO.sub.2 incubator, 
and the fused cells selected. After about 7-10 days cell supernatants of 
the fused cells are screened for antibody specific to the seminal vesicle 
marker of interest and the selected populations expanded. Hybridomas are 
picked and cultured. Monoclonal antibody is used directly or stored at 
-80.degree. C. in 0.5 ml aliquots. 
Example 5 - Identification and Isolation of Seminal Vesicle-Specific Marker 
Genes 
Polyclonal and monoclonal antibodies prepared in Examples 1, 2 and 4 are 
used to screen human seminal vesicle-specific cDNA expression libraries 
for seminal vesicle-specific marker proteins. Positive bacterial colonies 
or bacteriophage plagues are identified by Western blotting the 
supernatants of individual clones with antibody preparations. Positive 
clones are expanded and the recombinant DNA insert restriction mapped and 
sequenced using dideoxynucleotide chain termination methodology. These 
sequences are analyzed by computer alignment to available sequences in GEN 
BANK as in Example 3. DNA sequences of interest are subcloned into 
recombinant expression vectors for large scale production of seminal 
vesicle-specific marker. In addition, cDNA expression libraries are 
screened using .sup.32 P-radiolabeled oligonucleotides or DNA fragments as 
probes to either known genes, for example, semenogelin I, its three 
subunits, or semenogelin II, are prepared based on the peptide sequences 
obtained in Example 3. The oligonucleotide probes are synthesized to 
recognize peptide-encoding fragments enriched for the amino acid residues 
met, trp, phe, tyr, cys, his, gin, asn, lys, asp, and gin, in this order 
of priority. Such probes are capable of recognizing a minimum of 21 
deoxynucleotide residues corresponding to a minimum length of seven amino 
acid residues. 
Example 6 - Diagnostic Kits Containing Seminal Vesicle-Specific Marker. 
Microtiter plates are fixed with the seminal vesicle-specific marker made 
recombinantly as in Example 5 or purified conventionally as in Example 2. 
To the fixed antigen are added samples of blood or urine obtained from 
both normal healthy volunteers, patients with non-prostatic forms of 
cancer, and patients with suspected or confirmed cases of prostatic 
neoplasia with varying stages of severity. The samples are incubated for 
one hour at room temperature in a total volume of about 100 ul, after 
which, all liquid is removed by flicking the plates. Plates are washed 
three times with PBS, after which commercially available alkaline 
phosphatase conjugated anti-human antibodies are added to each well and 
the solution treated according to the appropriate visualization protocol. 
If the biological samples of, for example, blood or urine, contain seminal 
vesicle-specific antibodies, the antisera will bind to the fixed antigen. 
Positive indications are determined by comparing the binding observed with 
samples from the prostatic neoplasia positive patients with baseline 
determinations made for binding observed with samples obtained from normal 
individuals or individuals with other forms of cancer. 
Kits are also created using immuno-slot blots. Nitrocellulose membranes are 
placed in a slot blot apparatus and the various patient samples of serum, 
urine or semen, are placed individually in the slots and immobilized. 
Binding is detected using labeled seminal vesicle-specific marker or 
seminal vesicle-specific marker and a labeled secondary to detect bound 
marker and an appropriate visualization protocol. Alternatively, the 
marker protein may be bound to individual wells of the slot blot. Each 
well is cut with scissors to isolate immobilized antigen, immersed and 
incubated in patient samples, rinsed, and anti-human antibody conjugates 
applied to detect the complex. In addition to these methods, human 
antisera can be applied directly to Western blot membranes containing 
electrophoresed and transferred seminal vesicle components of homogenates 
or purified proteins. Following rinsing, anti-human antibody conjugates 
are applied to detect the complex. 
Example 7 - Diagnostic Kits Containing Antibody Specific To Seminal 
Vesicle-Specific Markers 
Microtiter plates are fixed with anti-seminal vesicle-specific rabbit 
polyclonal or murine monoclonal antibody prepared as described in Examples 
2 and 4, or human monoclonal antibody. Human monoclonal antibody is 
created by fusing human spleen cells, which were exposed to antigen, with 
human or partly human myeloma cells and selecting the appropriate 
hybridoma cells or by cloning the antibody binding site of a non-human 
antibody gene into the appropriate position of a human antibody expressing 
cell. To the fixed seminal vesicle-specific antibodies are added samples 
of blood, semen, or urine obtained from normal healthy volunteers, 
patients with non-prostatic forms of cancer, and patients with suspected 
and confirmed prostatic neoplasia with varying stages of severity. The 
plates are incubated for one hour at room temperature in a total volume of 
100 ul. After one hour, the liquids in the samples are removed by flicking 
the plates dry and the plates washed three times with PBS. 
Alkaline-phosphatase conjugated secondary antibody is added and the plates 
are treated according to the appropriate visualization protocol. Positive 
indications are determined by comparing the binding observed with the 
samples obtained from prostatic neoplasia patients with a baseline binding 
level observed in samples taken from normal healthy volunteers and 
patients with non-prostatic forms of cancer. 
Kits are also created using slot blots as in Example 6. Nitrocellulose 
membranes are placed in a slot blot apparatus and the various patient 
serum, urine, and semen samples, placed individually in the slots and 
immobilized. The amount of marker protein is determined using labeled 
seminal vesicle-specific antibody and an appropriate visualization 
protocol. In addition to these methods, the human samples are resolved on 
SDS PAGE gels and Western blotted using rabbit polyclonal, mouse 
monoclonal, or human monoclonal antibodies that recognize a specific 
marker protein. A secondary conjugated antibody is used to visualize the 
complex. 
Other embodiments or uses of the invention will be apparent to those 
skilled in the art from consideration of the specification and practice of 
the invention disclosed herein. It is intended that the specification and 
examples be considered exemplary only, with the true scope and spirit of 
the invention being indicated by the following claims.