WO2001001134A1 - A method for diagnosing selected adenocarcinomas - Google Patents

Abstract

A method for the early diagnosing of selected adenocarcinomas in a human comprising the steps of removing a bodily sample from the human, and assaying the bodily sample for elevated expression of a specific gene. The gene being assayed for in the bodily sample is the TGFB-4 gene (hereinafter referred to as the endometrial bleeding associated factor (ebaf) gene). The bodily sample can be tissue from a specific organ in the body, or a blood sample. Increased levels of ebaf in the sample relative to basal levels may be indicative of a mucinous adenocarcinoma of the colon or ovaries, or an adenocarcinoma of the testis.

Description

A Method for Diagnosing Selected Adenocarcinomas

CROSS REFERENCE

This application is a continuation-in-part (CIP) application of Serial No. 08/919,421 filed August 27, 1997, which is based on provisional application Serial No. 60/025,800, filed August 27, 1996, incorporated herein by reference .

GOVERNMENT SUPPORT

This invention was made with Government support under Grant Number CA46866 awarded by the National Institutes of Health (NIH) . The Government has certain rights in the invention.

FIELD OF INVENTION

A method for diagnosis of human bodily conditions, and more particularly, a method for diagnosing selected mucinous adenocarcinoma of the colon or ovaries, or an adenocarcinoma of the testis .

BACKGROUND OF INVENTION

Early diagnosis of particular pathological conditions of the human body can provide patients with adequate time to make well informed decisions regarding the treatment of their pathological condition, as well as prepare for the potential incapacitation of the patient. One such pathological condition is colon cancer. The American

Chemical Society has reported that colon cancer is the second most common cause of cancer in the United States

(Fleischer, D. et al . Detection and Survival of Colorectal Cancer (1989) JAMA 261(4) :580. It has been estimated that approximately 145,000 new cases of colon cancer are reported yearly in the United States, and the overall mortality rate of this pathological condition is almost 60%. Moreover, a diagnosis of colon cancer has been estimated to shorten that patient ' s life by six to seven years (Id) . Consequently, early detection of this pathological condition offers a patient the best hope of survival .

Approximately three principal screening tests for the early detection of colon cancer or precancerous polyps are presently available to physicians. One such test is the Fecal Occult Blood Test (FOBT) . Basically, this test is designed to test whether blood is present in the fecal material of the patient. The effectiveness of this test is dependent upon the assumptions that blood in the fecal material is indicative of the presence of colonic neoplasms, and that these neoplasms will bleed in sufficient quantity in order to cause a positive FOBT result . It is because of these necessary assumptions that applicants believe the FOBT contains significant shortcomings as a screening tool for colon cancer. For instance, it has been shown that not all colonic neoplasms bleed sufficiently into the colon. As a result, this test is readily capable of giving false negative results.

In addition, there are other factors which could result in a false positive result for this test . For example, it has been found that aspirin and other noninflammatory analgesics have been known to cause irritation in the stomach and increased gastro-intestinal tract blood loss, thereby producing in a false positive result . The patient ' s ingestion of rare beef and fruits and vegetables which contain catalases and peroxidases within 24 hours of administering the test may also cause a false positive result. Another screening test that is available is the Carcinoembryonic Antigen (CEA) test. CEA is a glycoprotein that may be produced by cancerous lesions in the colon. This test is designed to measure the concentration of CEA in the patient ' s blood to determine if it is elevated relative to normal levels . It is believed that an elevated level is due directly to the presence of colon cancer in the patient. It was hoped CEA would act as a genetic marker for colon cancer. Immunological techniques are usually used to measure CEA levels in the blood.

Soon after this test became available to health professionals, it was observed that this test was simply too insensitive to recognize numerous types of colon cancers . As a result, the CEA test was relegated to the detection of a recurrence of colon cancer after surgery is performed to remove cancerous lesions from the colon. Even in this role though, it has met with only limited success. In 1993, a study on the effectiveness of CEA testing in 1017 patients was published in the Journal of the American Medical Association. The study showed that 417 patients out of the original group developed recurrent colon cancer, and 247 of these had elevated CEA levels prior to diagnosis of recurrence. However, of the remaining 600 patients, 98 also had elevated CEA levels.

Hence the rate of false negatives for the test was 41%, and the rate of false-positive results was 16%. (Moertel,

C, eϋ al . An Evaluation of the Carcinoembryonic Antigen

(CEA) Test for moni toring Patients wi th resected Colon Cancer. JAMA 270 (8) : 954 (1993).

In concluding their study, the authors questioned the efficacy of the CEA Test. In support of this conclusion, they explained that, based on their data, the maximum anticipated gain from CEA monitoring would probably be a small number of lives saved (less than 1% of patients monitored) after resection and hepatic metastasis. In addition, the authors specifically stated, "Since the most defensible objective of CEA monitoring is detection of potentially resectable hepatic metastasis, it would also seem appropriate to consider alternative strategies that might fulfill this objective in a more sensitive, specific, and cost-effective manner." (Id)

Another method used to screen for colon cancer is to have the patient undergo a periodic sigmoidoscopic examination. The use of this screening test in a particular patient is dependent upon the age of the patient and whether he or she is a member of a high-risk population. Research on this screening technique has concluded this method to be the best known screening method for colon cancer presently available (see Selby, J. Sigmoidoscopy in the Periodic Health Examination of

Asymptomatic Adul ts JAMA (1989) 261(4) :595)

Researchers have also acknowledged that this screening method contains inherent limitations . For example, the high cost for the specialized instruments required to perform this screening test, and the special training required in the operation of the instruments in order to perform the procedure safely are acknowledged. Moreover, general patient discomfort while undergoing this screening is believed to be one of the obstacles in providing mass screening for the general population. Finally, health professionals acknowledge that there is a very slight risk of perforating a patient ' s colon while undergoing the procedure. Consequently, applicants believe a simple, cost effective screening test for colon cancer is needed.

Another type of pathological condition, present exclusively in women, is ovarian cancer. Ovarian cancer comes from cells of the ovary that grow and divide uncontrollably. Applicants believe that statistical information on ovarian cancer indicates that approximately one woman out of every fifty-five (approximately 1.8%) will develop ovarian cancer some time in her lifetime, and it was believed that in 1996, approximately 26,000 women would be diagnosed with ovarian cancer and approximately 14,500 women would die of the disease. Moreover, 85 to 90% of women diagnosed with the condition before it spreads from the ovary are cured. However, there is only a 20 to 25% chance of living after diagnosis, if the diagnosis is made after the disease has spread beyond the ovary.

Presently, there are methods available to diagnose ovarian cancer, but such methods have inherent limitations. One such method is assaying the patient's blood for elevated levels of Cancer Antigen 125 (CA 125) . It has been determined that eight out of ten women with advanced ovarian cancer, and in one out of two women with cancer localized in the ovary will have such elevated levels. However, endometriosis, pelvic inflammatory disease of the tubes and ovaries, uterine fibroids, and pregnancy can also elevate levels of CA 125 in the blood, resulting in false positives.

Another method involves screening the ovaries for a growth, surgically removing the growth, and then performing a biopsy on the growth. Screening can occur with a pelvic examination, during which the physician feels for growths on the ovary, or with special types of x-rays. If such a growth is discovered, it must be surgically removed, so that a biopsy can be performed. Another such screening method is ultrasound examination of the ovaries. Similar to the pelvic examination, this method provides no definitive answer regarding the presence of cancer in the ovaries .

Another pathological condition for which early diagnosis would benefit the patient is testicular cancer. With this type of cancer, the patient develops a growth within the body of the testicle. The physician must then determine whether the growth is cancerous using presently available diagnostic procedures .

One such procedure is to perform a biopsy on the growth through the scrotum. However, such a procedure presents a problem to the patient in that it could contaminate the scrotum, which could then be a site for the development of cancer. Moreover such a biopsy could disturb the pattern of nodal metastases, and make points for subsequent surgery difficult to predict .

Another such procedure is inguinal orechiectomy, which is done through an incision made above the inguinal ligament . The testicle is then brought up through the inguinal canal and examined visually. However this procedure has limitations in that it is done surgically, like the biopsy, and is a qualitative inspection of the testes. Consequently, a false positive or false negative can result from this procedure.

Another method available for diagnosing testicular cancer is assaying the patient ' s blood for elevated levels of Human Chorionic Gonadotrophin, beta subunit (Beta HCG) . However, this method contains inherent limitations in that it has been determined that low testosterone states, and marijuana use by the patient can produce false positives.

SUMMARY OF THE INVENTION

There is provided, in accordance with the present invention, a novel gene ebaf and the protein encoded by the same. Also provided are various sized mRNAs and isoforms encoding for the gene ebaf . There is also provided a method for the early diagnosing of selected adenocarcinomas in a human. Applicants believe that this method is accurate, dependable, inexpensive, and does not possess the shortcomings of the prior art as explained above. In particular, the present invention describes a method for diagnosing an adenocarcinoma in a human comprising the steps of removing a bodily sample, including various bodily fluids, from the human, and assaying the bodily sample for elevated expression of a specific gene. The bodily sample can be either tissue from a particular organ, such as the colon or the ovary, a sample of blood, urine, saliva, normal and abnormal bodily fluids including peritoneal, pericardial, pleura, senovial and cerebrospinal fluid, seminal plasma, ovarian follicular fluid, peritoneal fluid, milk, etc. For purposes of this application, "expression" means either the transcription of the specific gene into at least one mRNA transcript, or the translation of at least one mRNA into a protein.

The specific gene referred to above is the TGFB-4 gene (hereinafter referred to as the endometrial bleeding associated factor ( ebaf) gene) . Applicants recently discovered this gene in humans (see Ravi Kothapalli, Ibrahim Buyuksal, Shi-Qi Wu, Nasser Chegini, Siamak Tabibzadeh: Detection of ebaf. a novel human gene of the

TGF-β super family; association of gene expression wi th endometrial bleeding J. Clin. Invest. 1997, 99:2342-2350, which is hereby incorporated by reference herein) . The cDNA sequence of the ebaf gene is set forth in SEQ. ID NO. 1.

Applicants have also discovered that, due to alternative splicing during the transcription of the gene into mRNA, three different mRNA transcripts can result from the transcription of the ebaf gene. One of the transcripts is 1.5 kb in size, one is 2.1 kb, and the remaining is 2.5 kb. Consequently, such processing will produce three isoforms upon translation of the transcripts. Regardless, elevated expression of the ebaf gene can be determined from elevated levels of any transcript or any isoform. Hence, one object of the present invention is to provide an accurate, reliable method for the diagnosis and detection of an adenocarcinoma of the testis, or a mucinous adenocarcinoma of the colon in a human male.

Another object of the present invention is to provide an accurate, reliable method for the diagnosis and detection of a mucinous adenocarcinoma of the ovaries, or a mucinous adenocarcinoma of the colon in a human female .

Yet another object of the present invention is to provide a method of diagnosing and detecting a mucinous adenocarcinoma of the ovaries or colon in a female human, or a mucinous adenocarcinoma of the colon or an adenocarcinoma of the testis in a human male that is selective for such adenocarcinomas . While the ebaf gene disclosed in the present is expressed in the adenocarcinomas mentioned above, it is not expressed in other types of adenocarcinomas, such as Squamous Cell Carcinoma (SCO , lymphoma or adenocarcinoma. Consequently, the present invention is very selective for the type of adenocarcinomas it is designed to detect. Moreover, in normal tissues, the ebaf gene is expressed only the ovary, pancreas, rectum, endometrium immediately prior to and during the menstrual cycle, and weakly in the colon and the kidney. Consequently, the number of false positive resulting from the use of the present invention is limited.

Yet still another object of the present invention is to provide a test for adenocarcinomas of the testis, and mucinous adenocarcinomas of the colon and ovaries . As stated above, only six organs are presently known to express the ebaf gene constitutively. Applicants believe this constitutive expression results in a basal level of expression of the ejbaf gene in the blood, urine, saliva and other bodily fluids. However, if increased levels of expression of the ebaf^" gene are detected in the fluid of a human relative to the basal level, they indicate the presence of an adenocarcinoma of the testis, or a mucinous adenocarcinoma of the colon or ovary. For example, if increased levels of expression of the ejbaf gene are detected in a fluid sample from a human male, such levels are indicative of an adenocarcinoma of the testis or a mucinous adenocarcinoma of the colon. If increased levels of expression of the ebaf gene are detected in a sample of fluid taken from a female after her period, then such increased levels may be indicative of the presence of a mucinous adenocarcinoma in the colon or ovaries, provided the female does not suffer from abnormal uterine bleeding.

DESCRIPTION OF FIGURES

Figure 1 is a Northern blot analysis of ebaf mRNA in normal tissues; Figure 2 is a Northern blot analysis of ebaf mRNA in colonic adenocarcinomas;

Figure 3 is a Northern blot analysis of ebaf mRNA in testicular cancers;

Figure 4 is a photograph showing the demonstration of the eJbaf in the serum, saliva and urine; serum samples were obtained from a normal fertile woman throughout the menstrual cycle days 3, 11, 18 and 25 and the first day of menstrual bleeding (Dl) ; on the first day of menstrual bleeding, the saliva as was as urine was also obtained, the extracted proteins were subjected to protein gel electrophoresis; the proteins on the gel were transferred to a Nitrocellulose membrane and stained with the antibody to ebaf; the 31 kD of the ebaf protein is found on the first day of menstruation in the blood, saliva and urine;

Figure 5 is photograph showing a demonstration of immunoreactivity and specificity of the C-terminal rabbit anti-serum to ebaf by Western blot analysis; A (left) : In each lane, 10 mg of extracted endometrial proteins was resolved in a 15% gel by SDS-PAGE and then subjected to Western blot analysis; the blot was probed with the C- terminal anti-serum alone _.(left lane) and with the antiserum-preincubated with a 100 molar excess of the CASDGALVPRRLQHRP-amide (SEQ. ID NO. 4); the antiserum was used at 1:250 dilution of a solution containing 0.8 mg protein/ml; the peptide was used at 0.32 mg/ml; several immunoreactive bands ("55/60, ~41, ~32, ~26 kD) were detected by the antiserum (left lane) ; immunoreactivity of the antiserum was markedly reduced by its pre-incubation with the peptide (right lane) ; B (right) : 250 mg total protein from a menstrual day 1 endometrium was immunoprecipiated using the rabbit antiserum to ebaf; the detected protein bands were similar in size to those identified by Western blotting; in addition, an additional, ~11 kD, band was also detected; the size of the proteins is shown in kilodalton; Figure 6 is a photograph showing Western blot analysis of the ebaf in the lysates and conditioned media of cells transfected with full length ebaf cDNA; the conditioned media of ebaf cDNA transfected cells (lanes 1 and 3) , their cell lysates (lanes 2 and 4) and recombinant E-coli produced ebaf (5 ng, lanes 5 and 6) were subjected to Western blot analysis and probed with affinity purified rabbit polyclonal C-terminal antibody to ebaf (lanes 1-5) and a monoclonal C-terminal antibody to ebaf (lane 6) ; lanes 1 and 2; NIH-3T3 cells transfected with the anti-sense ebaf cDNA prepared from the coding region of ebaf; lanes 3 and 4 : NIH-3T3 cells transfected with the sense cDNA prepared from the coding region of ebaf; lanes 5 and 6: recombinant 26 kD E-coli produced ebaf; size is shown in kD;

Figure 7 is a photograph showing immunoreactive ebaf bands in the endometria of infertile women; in each lane, 10 mg of extracted endometrial proteins of each endometrium was resolved in a 15% gel by SDS-PAGE and then subjected to Western blot analysis; the blots were probed with the affinity purified rabbit C-terminal anti- serum to ebaf; the anti-serum reacts with the protein bands in the size of ~55/60, ~41, ~32 and ~26 kD; lane 1: endometrial proteins from a menstrual day 1 endometrium; lanes 1-2: normal endometria from cycle days 20-21; lanes 3-23: Endometria of infertile patients; the size of the proteins is shown in kilodalton; different species of the ebaf protein are relatively more abundant in different patients (endometriosis) ; and

Figure 8 are depictions showing: tagging ebaf protein; ebaf was tagged as shown; B: The sequence of several clones with tags at the carboxy terminal of ebaf

(upper panel) , after the RGKR site (middle panel) and the

RHGR site (lower panel) are shown; tags are boxed and an inserted restriction enzyme site, BamHl, and the RGKR and

RHGR sites are marked; C: In a Western blot analysis, the monoclonal antibody to HA tag detects the tagged ebaf in the cell lysate (precursor) (lane 1) and conditioned medium (lane 2) (32 kD processed form) of the 293T cells transfected with the ebaf cDNA tagged at the first RGKR sites .

DESCRIPTION OF THE INVENTION

Generally, the present invention provides the nucleic acid sequence encoding for endometrial bleeding associated factor (ebaf) having the nucleic acid sequence shown in SEQ. ID NO. 1 and mRNA splice variants. also provided is the predicted amino acid sequence of ebaf protein SEQ. ID NO. 5. Furthermore, peptide epitopes from ebaf protein are provided, particularly for generating antibodies to ebaf. More specifically, the present invention provides a diagnostic tool and method for determining the presence of an adenocarcinoma by screening a bodily sample, particularly a fluid sample, for the presence of ebaf or the mRNA encoding ebaf. In a particular embodiment of the invention, applicants used the Northern Blot method to determine the expression of the ebaf gene in particular bodily samples. However, as stated above, any known method capable of detecting whether levels of expression of the ebaf gene in a bodily sample are elevated relative to levels observed in a normal bodily sample of the same type is an acceptable method of practicing the invention. Such assays include, but are not limited to: collecting proteins from a bodily sample and performing an immunoassay on the proteins using monoclonal or polyclonal antibodies raised against the isoforms produced from the expression of the e af gene;

collecting DNA from a bodily sample and performing a southern blot on the DNA using a probe that is complimentary to all or a portion of the ebaf cDNA;

collecting RNA from a bodily sample, reverse transcribing the RNA into DNA (RT) , amplifying the DNA (via PCR) , and then performing a southern blot on the DNA using a probe that is complimentary to all or a portion of the ebaf cDNA. The ebaf is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, and other factors known to medical practitioners. The "effective amount" for purposes herein is thus determined by such considerations as are known in the art .

In the method of the present invention, the ebaf can be administered in various ways . It should be noted that the e af can be administered alone or in combination with pharmaceutically acceptable carriers. The compounds can be administered orally or parenterally. Implants of the compounds are also useful. Gene therapy, as used herein, refers to the transfer of genetic material (i.e., DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition phenotype . The genetic material of interest encodes a product (i.e., a protein, polypeptide, peptide, functional RNA, antisense) whose production in vivo is desired. For example, the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value as shown in the gene associated with human infertility herein above. Alternatively, the genetic material of interest encodes a suicide gene . Standard gene therapy techniques known in the art and not specifically described are generally followed as in "Gene Therapy" (Advances in Pharmacology 40, Academic Press, 1997) . Two basic approaches to gene therapy have evolved:

(1) ex vivo and (2) in vivo gene therapy. In ex vivo gene therapy cells are removed from a patient, and while being cultured are treated in vi tro . Generally, a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient. These genetically reimplanted cells have been shown to express the transfected genetic material in si tu .

Standard molecular biology techniques known in the art and not specifically described are generally followed as in Sambrook et al . , Molecular Cloning: A Laboratory Manual , Cold Springs Harbor -Laboratory, New York (1989, 1992), and in Ausubel et al . , Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989) . Additionally, standard methods in immunology known in the art and not specifically described are generally followed as in Stites et al.

(eds) , Basic and Clinical Immunology (8^th Edi tion) ,

Appleton & Lange, Norwalk, CT (1994) and Mishell and

Shiigi (eds) , Selected Methods in Cellular Immunology,

W.H. Freeman and Co., New York (1980). Cloning techniques are provided by the present invention as are commonly known in the art and not specifically described are generally followed as in Sambrook et al . , Molecular Cloning: A Laboratory Manual , Cold Springs Harbor Laboratory, New York (1989, 1992), and in Ausubel et al . , Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989) .

Immunoassays are also provided by the present invention. In general, ELISAs are the preferred immunoassays employed to assess a specimen. Both polyclonal and moloclonal antibodies can be used in the assays . The specific assay to be used can be determined by one skilled in the art .

Antibody production is provided by the present invention. Antibodies may be prepared against the immunogen, or any portion thereof, for example a synthetic peptide based on the sequence, for example CASDGALVPRRLQHRP-amides . As stated above, antibodies are used in assays and are therefore used in determining if the appropriate protein has been identified. Antibodies can also be used for removing enzymes from red cell suspensions after enzymatic conversion. Immunogens can be used to produce antibodies by standard antibody production technology well-known to those skilled in the art as described generally in Harlow and Lane, Antibodies : A Laboratory Manual , Cold Springs Harbor Laboratory, Cold Spring Harbor, NY, 1988 and Borrebaeck, Antibody Engineering - A Practical Guide, W.H. Freeman and Co. , 1992. By "bodily sample" as it is used throughout this application, it is meant any bodily fluids which can be removed from the body, for example, but not limited to, normal and abnormal bodily fluids including peritoneal, pericardial, pleura, senovial and cerebrospinal fluid, urine, saliva, seminal plasma, ovarian follicular fluid, peritoneal fluid, milk, etc.

In another embodiment of the present invention there is provided a vector which contains the isolated nucleic acid molecule having a nucleic acid sequence as shown in SEQ. ID NO. 1. More specifically, the preferred vector is a plasmid. Additionally, there is provided a host vector system for the production of a polypeptide, in which new polypeptide encoded for is endometrial bleeding factor which contains a vector and suitable host cell . The suitable host cell may be a bacterial cell, insect cell or mammalian cell, as can be determined by one skilled in the art for the specific function.

Also provided is a method of producing a polypeptide which is endometrial bleeding associated factor which includes growing the host cells described above, under suitable conditions, thus permitting production of the polypeptide and then recovering the polypeptide produced. The conditions under which the host cells are grown are readily known by people of skill in the art. Also provided is a diagnostic tool for determining the presence of an adenocarcinoma in a human including the steps of removing a bodily sample from the human and assaying the bodily sample for elevated expression of a specific gene. The bodily sample can include, but is not limited to, bodily fluids including, but not limited to, bodily tissues, for example, but not limited to, normal and abnormal bodily fluids including peritoneal, pericardial, pleura, senovial and cerebrospinal fluid, urine, saliva, seminal plasma, ovarian follicular fluid, peritoneal fluid, milk, etc. Additionally, the diagnostic tool can be used for screening for elevated levels of the mRNA transcript encoding for ebaf . Such screening methods can include, but are not limited to, Northern blot analysis, Western blot analysis, immunohistochemical staining, and PCR.

Additionally, several monoclonal and polyclonal antibodies have been raised to human ebaf peptides. Using these antibodies, ebaf can be detected in bodily samples.

I. Northern Blotting

The RNA was extracted from samples by using acid guanidinium thiocyanate-phenol-chloroform extraction method as described in Chomczynski, P. and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal . Biochem.

1987, 162:156-159 which is hereby incorporated by reference herein. The tissues samples were homogenized in

"RNA STAT-60" reagent. Each 50-100 mg of tissue was homogenized in 1 ml of "RNA STAT-60" reagent in a glass or Teflon Dounce homogenizer. Each homogenate was stored for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes. Then, 0.2 ml of chloroform was added for each ml of "RNA STAT-60" reagent used. Each sample was covered and shaken vigorously for 15 seconds and allowed to stand at room temperature for 2-3 minutes. Following centrifugation at 12,000 xg for 15 minutes at 4° C, each homogenate was separated into a lower phenol/chloroform phase and an upper aqueous phase. RNA in the upper aqueous phase was transferred to fresh tubes and mixed with isopropoanol to precipitate the total RNA. After centrifugation and drying, the precipitated RNA was dissolved in diethylpyrocarbonate (DEPC) -treated water by vigorous pipetting and by a gentle heating at 55-60°C. The amount of RNA in each sample was determined spectro- photometrically and its quality was evaluated by the integrity of ribosomal RNA by electrophoresis of 20 mg of total RNA in 1% formaldehyde-agarose gel in the presence of ethidium bromide. Northern blotting was then performed. Briefly, 20 mg of total RNA of each sample was denatured at 65°C in an RNA loading buffer, electrophoresed in 1% agarose containing 2.2 M formaldehyde gel, and blotted onto a "HYBOND" nylon membrane using a positive pressure transfer apparatus (Posiblot, Stratagene, La Jolla, CA) . The RNA was fixed to the membrane by UV cross-linking. Using a "PRIME-A-GENE" kit, cDNA was labeled with [³P] to a high specific activity, and purified by Nick columns. Membranes were prehybridized in 50% formamide, lOx Denhardt's solution, 4% saline sodium citrate (SSC) , 0.05 M sodium pyrophosphate and 0.1 mg/ml of denatured Hering sperm DNA at 42°C for 2-4 hr and hybridized for 16 hours at 42°C with 10^e cpm/ml of heat-denatured probe in the same buffer containing 10% dextran sulfate. Then, membranes were sequentially washed three times in 4x SSC, one time in 0.5x SSC and then one time in O.lx SSC. All washes contained 0.1% sodium dodecyl sulfate (SDS) , and were done at 65°C for 20 minutes each. The membranes were subjected to autoradiography at -70°C with intensifying screens. The same blot was stripped and reprobed for GAPDH. To reprobe a blot, the probe was stripped from the membrane in 75% formamide, O.lx saline sodium phosphate ETDA (SSPE) , and 0.2% SDS at 50°C for one hour.

Figure 1 shows the Northern Blot of ebaf mRNA in normal tissues. In FIG. 1, 20 mg total RNA from each tissue (lane 1: normal menstrual endometrium serving as the positive control and normal tissues (lane 2: spleen, lane 3: lymph node, lanes 4 and 5: stomach, lane 6: lung, lane 8: liver, lane 9 and 10: ovary, lane 11: rectum, lane 12: testis, lane 13: pancreas) was subjected to the Northern blot analysis using the entire eJbaf cDNA (SEQ. ID NO. 1) as the probe. As shown, a band of e af mRNA in the size of 2.5 kilobase (kb) is detected in the endometrium. A weak 2.1 kb ebaf mRNA is detected in the ovary, rectum, and testis. In the pancreas, both the 2.1 and 2.5 kb e af mRNA is detected. The ebaf gene was not expressed in the breast, stomach, small bowel, colon, kidney, lung, fallopian tube, spleen and lymph node. Table 1 displays these results in tabular form:

Table 1. Expression of ebaf mRNA in Normal Tissues

signal not detected of eleven samples tested, only one showed a strong signal. The remaining were not apparent.

The expression of the ejbaf gene was then examined in cancers derived from cells of different lineages. In eleven adenocarcinomas of colon, adjacent normal colonic tissues, non-involved by the tumor were available for the study. The RNAs from the neoplastic and surrounding normal tissues were both subjected to the Northern blot analysis for the detection of the ebaf mRNA.

Applicants also examined a host of other types of cancer for the expression of the ejbaf gene. Using the same Northern Blot procedure stated in each of the previous examples, applicants collected the following data regarding the expression of the ebaf gene in non-mucinous adenocarcinomas : Table 2. Expression of ebaf mRNA in the Non-Mucinous Adenocarcinomas

Normal tissues around the tumors were available for the Northern blot analysis and did not exhibit eJbaf mRNA

: Signal not detected Squamous cell carcinomas and non-epithelial tumors for the expression were also examined for expression of the ebaf gene The same Northern Blot protocol as explained above was also used for these tumors. The results of these tests are shown in Tables 3 and 4, respectively.

Table 3 » Expression of eJbaf mRNA in Squamous Cell Carcinomas

Signal not detected

Table 4. Expression of ebqfmRNA in Non-epithelial Tumors

- : Signal not detected

The above discussion provides a factual basis for the use of ebaf as a diagnostic tool . The method used with a utility of the present invention can be shown by the following non-limiting examples and accompanying figures .

EXAMPLES

General Methods: The following Methods and Examples included herewith and incorporated by reference in their entirety further show the invention.

Methods: General methods in molecular biology: Standard molecular biology techniques known in the art and not specifically described are generally followed as in Sambrook et al . , Molecular Cloning: A Laboratory Manual , Cold Springs Harbor Laboratory, New York (1989, 1992), and in Ausubel et al . , Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989) . Polymerase chain reaction (PCR) is carried out generally as in PCR Protocols : A Guide To Methods And Applications, Academic Press, San Diego, CA (1990) . Reactions and manipulations involving other nucleic acid techniques, unless stated otherwise, are performed as generally described in Sambrook et al . , 1989, Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press, and methodology as set forth in United States patents 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 and incorporated herein by reference. In-situ (In-cell) PCR in combination with Flow Cytometry can be used for detection of cells containing specific DNA and mRNA sequences (Testoni et al, 1996, Blood 87:3822.)

General methods in immunology: Standard methods in immunology known in the art and not specifically described are generally followed as in Stites et al.(eds), Basic and Clinical Immunology (8th Edition), Appleton & Lange, Norwalk, CT (1994) and Mishell and Shiigi (eds) , Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York (1980).

Immunoassays: In general, ELISAs are one type of immunoassay employed to assess a specimen in the method of the present invention. ELISA assays are well known to those skilled in the art. Both polyclonal and monoclonal antibodies can be used in the assays . Where appropriate other immunoassays, such as radioimmunoassays (RIA) can be used as are known to those in the art . Available immunoassays are extensively described in the patent and scientific literature. See, for example, United States patents 3,791,932; 3,839,153; 3,850,752; 3,850,578 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219 5,011,771 and 5,281,521 as well as Sambrook et al, Molecular Cloning: A Laboratory Manual , Cold Springs Harbor, New York, 1989 Antibody Production: Antibodies as controls and for use in the immunoassays of the present invention as for example control antibodies can be either monoclonal, polyclonal or recombinant. Conveniently, the antibodies may be prepared against the immunogen or portion thereof for example a synthetic peptide based on the sequence, or prepared recombinantly by cloning techniques or the natural gene product and/or portions thereof may be isolated and used as the immunogen. Immunogens can be used to produce antibodies by standard antibody production technology well known to those skilled in the art as described generally in Harlow and Lane, Antibodies : A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988 and Borrebaeck, Antibody Engineering - A Practical Guide, W.H. Freeman and Co., 1992. Antibody fragments may also be prepared from the antibodies and include Fab, F(ab')₂, and Fv by methods known to those skilled in the art.

For producing polyclonal antibodies a host, such as a rabbit or goat, is immunized with the immunogen or immunogen fragment, generally with an adjuvant and, if necessary, coupled to a carrier; antibodies to the immunogen are collected from the sera. Further, the polyclonal antibody can be absorbed such that it is monospecific. That is, the sera can be absorbed against related immunogens so that no cross-reactive antibodies remain in the sera rendering it monospecific.

For producing monoclonal antibodies the technique involves hyperimmunization of an appropriate donor with the immunogen, generally a mouse, and isolation of splenic antibody producing cells. These cells are fused to a cell having immortality, such as a myeloma cell, to provide a fused cell hybrid which has immortality and secretes the required antibody. The cells are then cultured, in bulk, and the monoclonal antibodies harvested from the culture media for use .

For producing recombinant antibody (see generally Huston et al, 1991; Johnson and Bird, 1991; Mernaugh and Mernaugh, 1995) , messenger RNAs from antibody producing B-lymphocytes of animals, or hybridoma are reverse- transcribed to obtain complimentary DNAs (cDNAs) . Antibody cDNA, which can be full or partial length, is amplified and cloned into a phage or a plasmid. The cDNA can be a partial length of heavy and light chain cDNA, separated or connected by a_, linker. The antibody, or antibody fragment, is expressed using a suitable expression system to obtain recombinant antibody. Antibody cDNA can also be obtained by screening pertinent expression libraries. The antibody can be bound to a solid support substrate or conjugated with a detectable moiety or be both bound and conjugated as is well known in the art.

(For a general discussion of conjugation of fluorescent or enzymatic moieties see Johnstone & Thorpe, Immuno chemistry in Practice, Blackwell Scientific Publications, Oxford, 1982.) The binding of antibodies to a solid support substrate is also well known in the art. (see for a general discussion Harlow & Lane Antibodies : A Laboratory Manual , Cold Spring Harbor Laboratory Publications, New York, 1988 and Borrebaeck, Antibody Engineering - A Practical Guide, W.H. Freeman and Co., 1992) The detectable moieties contemplated with the present invention can include, but are not limited to, fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, b-galactosidase, peroxidase, urease, fluorεscein, rhodamine, tritium, ¹⁴C and iodination. Recombinant Protein Purification Marshak et al , "Strategies for Protein Purification and Characterization. A laboratory course manual." CSHL Press, 1996.

Transgenic and Knockout Methods : The present invention may provide for transgenic gene and polymorphic gene animal and cellular (cell lines) models as well as for knockout models wherein the gene associated with human female infertility is _ either inserted and/or the corresponding animal gene "knocked out". These models can, for example, be used for the study of therapeutics for treating infertility. These models are constructed using standard methods known in the art and as set forth in United States Patents 5,487,992, 5,464,764, 5,387,742,

5,360,735, 5,347,075, 5,298,422, 5,288,846, 5,221,778,

5,175,385, 5,175,384,5,175,383, 4,736,866 as well as

Burke and Olson (1991), Capecchi (1989), Davies et al .

(1992), Dickinson et al . (1993), Duff and Lincoln (1995), Huxley et al . (1991), Jakobovits et al . (1993), Lamb et al. (1993), Pearson and Choi (1993), Rothstein (1991), Schedl et al. (1993), Strauss et al . (1993). Further, patent applications WO 94/23049, WO 93/14200, WO 94/06908, WO 94/28123 also provide information. For gene therapy: By gene therapy as used herein refers to the transfer of genetic material (i.e., DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition phenotype . The genetic material of interest encodes a product (i.e., a protein, polypeptide, peptide, functional RNA, antisense) whose production in vivo is desired. For example, the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value as shown in the gene associated with human infertility herein above. Alternatively, the genetic material of interest encodes a suicide gene. For a review see, in general, the text "Gene Therapy" (Advances in Pharmacology 40, Academic Press, 1997) .

Two basic approaches to gene therapy have evolved: (1) ex vivo and (2) in vivo gene therapy. In ex vivo gene therapy cells are removed from a patient, and while being cultured are treated in vi tro. Generally, a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method (transfection, transduction, - homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient. These genetically reimplanted cells have been shown to express the transfected genetic material in situ.

In in vivo gene therapy, target cells are not removed from the subject rather the genetic material to be transferred is introduced into the cells of the recipient organism in si tu, that is within the recipient. In an alternative embodiment, if the host gene is defective, the gene is repaired in si tu [Culver, 1998] . These genetically altered cells have been shown to express the transfected genetic material in si tu .

The gene expression vehicle is capable of delivery/ transfer of heterologous nucleic acid into a host cell . The expression vehicle may include elements to control targeting, expression and transcription of the nucleic acid in a cell selective manner as is known in the art . It should be noted that often the 5 'UTR and/or 3 'UTR of the gene may be replaced by the 5 ' UTR and/or 3 'UTR of the expression vehicle. Therefore as used herein the expression vehicle may, as needed, not include the 5 'UTR and/or 3 'UTR of the actual gene to be transferred and only include the specific amino acid coding region. The expression vehicle can include a promoter for controlling transcription of the heterologous material and can be either a constitutive or inducible promoter to allow selective transcription. Enhancers that may be required to obtain necessary transcription levels can optionally be included. Enhancers are generally any non- translated DNA sequence which works contiguously with the coding sequence (in cis) to change the basal transcription level dictated by the promoter. The expression vehicle can also include a selection gene as described herein below.

Vectors can be introduced into cells or tissues by any one of a variety of known methods within the art . Such methods can be found generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual , Cold Springs Harbor Laboratory, New York (1989, 1992) , in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989) , Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, MI (1995), Vega et al . , Gene Targeting, CRC Press, Ann Arbor, MI (1995) , Vectors : A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA (1988) and Gilboa et al (1986) and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see United States patent 4,866,042 for vectors involving the central nervous system and also United States patents 5,464,764 and 5,487,992 for positive- negative selection methods. Introduction of nucleic acids by infection offers several advantages over the other listed methods . Higher efficiency can be obtained due to their infectious nature. Moreover, viruses are very specialized and typically infect and propagate in specific cell types . Thus, their natural specificity can be used to target the vectors to specific cell types in vivo or within a tissue or mixed culture of cells. Viral vectors can also be modified with specific receptors or ligands to alter target specificity through receptor mediated events . A specific example of DNA viral vector for introducing and expressing recombinant sequences is the adenovirus derived vector Adenop53TK. This vector expresses a herpes virus thymidine kinase (TK) gene for either positive or negative selection and an expression cassette for desired recombinant sequences. This vector can be used to infect cells that have an adenovirus receptor which includes most cancers of epithelial origin as well as others. This vector as well as others that exhibit similar desired functions can be used to treat a mixed population of cells and can include, for example, an in vi tro or ex vivo culture of cells, a tissue or a human subject.

Additional features can be added to the vector to ensure its safety and/or enhance its therapeutic efficacy. Such features include, for example, markers that can be used to negatively select against cells infected with the recombinant virus . An example of such a negative selection marker is the TK gene described above that confers sensitivity to the antibiotic gancyclovir. Negative selection is therefore a means by which infection can be controlled because it provides inducible suicide through the addition of antibiotic. Such protection ensures that if, for example, mutations arise that produce altered forms of the viral vector or recombinant sequence, cellular transformation will not occur.

Features that limit expression to particular cell types can also be included. Such features include, for example, promoter and regulatory elements that are specific for the desired cell type. In addition, recombinant viral vectors are useful for in vivo expression of a desired nucleic acid because they offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells . The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. . This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny. Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

As described above, viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types. The targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell . The vector to be used in the methods of the invention will depend on desired cell type to be targeted and will be known to those skilled in the art . For example, if breast cancer is to be treated then a vector specific for such epithelial cells would be used. Likewise, if diseases or pathological conditions of the hematopoietic system are to be treated, then a viral vector that is specific for blood cells and their precursors, preferably for the specific type of hematopoietic cell, would be used. Retroviral vectors can be constructed to function either as infectious particles or to undergo only a single initial round of infection. In the former case, the genome of the virus is modified so that it maintains all the necessary genes, regulatory sequences and packaging signals to synthesize new viral proteins and RNA. Once these molecules are synthesized, the host cell packages the RNA into new viral particles which are capable of undergoing further rounds of infection. The vector's genome is also engineered to encode and express the desired recombinant gene. In the case of non- infectious viral vectors, the vector genome is usually mutated to destroy the viral packaging signal that is required to encapsulate the RNA into viral particles. Without such a signal, any particles that are formed will not contain a genome and therefore cannot proceed through subsequent rounds of infection. The specific type of vector will depend upon the intended application. The actual vectors are also known and readily available within the art or can be constructed by one skilled in the art using well-known methodology.

The recombinant vector can be administered in several ways. If viral vectors are used, for example, the procedure can take advantage of their target specificity and consequently, do not have to be administered locally at the diseased site. However, local administration can provide a quicker and more effective treatment, administration can also be performed by, for example, intravenous or subcutaneous injection into the subject. Injection of the viral vectors into a spinal fluid can also be used as a mode of administration, especially in the case of neuro- degenerative diseases. Following injection, theviral vectors will circulate until they recognize host cells with the appropriate target specificity for infection. An alternate mode of administration can be by direct inoculation locally at the site of the disease or pathological condition or by inoculation into the vascular system supplying the site with nutrients or into the spinal fluid. Local administration is advantageous because there is no dilution effect and, therefore, a smaller dose is required to achieve expression in a majority of the targeted cells. Additionally, local inoculation can alleviate the targeting requirement required with other forms _of administration since a vector can be used that infects all cells in the inoculated area. If expression is desired in only a specific subset of cells within the inoculated area, then promoter and regulatory elements that are specific for the desired subset can be used to accomplish this goal. Such non-targeting vectors can be, for example, viral vectors, viral genome, plasmids, phagemids and the like. Transfection vehicles such as liposomes can also be used to introduce the non-viral vectors described above into recipient cells within the inoculated area. Such transfection vehicles are known by one skilled within the art.

Delivery of gene products/therapeutics is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners. The pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms, i.e. infertility, and other indicators as are selected as appropriate measures by those skilled in the art.

Example 1 % Detection of Mucinous Adenocarcinomas of the Colon

In Figure 2, 20 mg total RNA from a normal late secretory endometrium which served as the positive control (lane 1) as well as mucinous adenocarcinomas of colon (lanes 2,4,6), non-mucinous adenocarcinomas of colon (lanes 8, 10, and 12) and adjacent normal colon (lanes 3, 5, 7, 9, 11 and 13) was subjected to the Northern blot analysis using the entire placental-derived ebaf cDNA as the probe (upper panel). Here, the expression of a 2.1 kb ebaf mRNA was detected in seven of the eleven cases of adenocarcinomas of the colon. The histological evaluation of the positive cases revealed them to have a mucinous differentiation.

Example 2 % Detection of Adenocarcinomas of the Testis

Figure 3 is a Northern blot displaying the results of an examination of five cases of testicular cancer for the expression of ebaf gene. 20 mg total RNA from a normal menstrual endometrium which served as the positive control

(lane 1) and each tumor tissue (lane 2: teratoma- embryonal cell carcinoma, lane 3: mixed germ cell tumor containing embryonal carcinoma, lanes 4-6; seminoma) were subjected to the Northern blot analysis using the entire placental-derived eJbaf cDNA as the probe (upper panel) .

The blot was exposed for long duration to detect ebaf mRNA in the neoplastic tissues. This resulted in the overexposure of the ebaf mRNAs detected in the endometrium. The results shown in Figure 3 indicate that a 2.5 kb ebaf mRNA is detected in the tumors containing embryonal carcinoma. The 2.1 kb mRNA is also detected in two out of three cases of seminoma. The integrity of RNA and equal loading was verified by staining the 18S and 28S ribosomal RNAs (not shown) and hybridization of the blots with a cDNA probe to GAPDH (lower panel) .

11₌ RT-PCR of RNA in the Sample Followed by Southern Blotting

Cells in the bodily sample should be lysed in 0.8 ml of "TRIREAGENT" solution (MRC Inc, Cincinnati, OH) in the presence of glycogen carrier. Supernatant containing RNA should be combined with 0.2 ml of chloroform, precipitated with isopropanol and washed with 70% ethanol. The RNA pellet should then be dissolved in RNAse-free water and incubated at 37° C with 40 U DNAse I (Gibco-BRL Life Technologies) for 30 minutes. The reaction would be terminated by the addition of EDTA (20 mM) and incubation for 10 min at 65° C. Total RNA should then be precipitated overnight at -80° C by the addition of three volumes of absolute ethanol-sodium chloride mixture. The quantity of the RNA would then be determined spectrophotometrically.

The total RNA would then be reverse transcribed in a 20 ml volume containing 2 mg RNA; 0.2 mg oligo (dT) , 1.25 mM of each of dATP, dCTP, dGTP, dTTP; 5 U AMV reverse transcriptase; 10 mM MeHgOH, 88 mM β-mercaptoethanol; 10 U RNAsin; 100 mM Tris-HCl (pH 8.3); 40 mM KCl and 10 mM MgCl2. After 60 minutes of incubation at 42° C, the reaction mixture would be heated to 95° C for 3 minutes. Following addition of 5 U of AMV, the reverse transcription would be carried out for an additional 60 minutes. After a final incubation at 95° C for 3 minutes, reverse transcription would be terminated by placing the reaction mixture at 0° C.

1 mg of reverse transcribed RNA would then be amplified with 0.5-1 mM of each of the 5' and 3' primers specific for IL-10 in a 50 ml reaction volume containing 1.25 U AmpliTaq DNA polymerase, 1.25 mM MgCl₂, 20 mM of each of dATP, dCTP, dGTP, dTTP, 10 mM Tris-HCl (pH 8.3), 50 mM KC1, and sterile distilled water. Negative control tubes would receive non-reverse transcribed RNA to verify absence of contaminating DNA..Positive control tubes would receive all the reagents in the reaction mixture, however, the primers used would be specific for β-actin. The reaction mixture would be overlayered with 50 ml of mineral oil and the tubes would be heated for 5 minutes at 95° C. After initiation of temperature cycling with a Dual- Block Thermal Cycler (Ericomp, San Diego, CA) , samples would be amplified for 35 cycles. The denaturation temperature would be 95° C for 1 minute, annealing temperature would be 55° C for 1 minute and the extension temperature would be 72° C for 2.5 minutes . Temperature cycling would be concluded with a final extension at 72° C for 10 minutes and the reaction products would be maintained at 4° C. Amplified products will be resolved in a 2% agarose gel and the bands would be visualized by ethidium bromide staining. The fX174 Hae III RF DNA fragments and the 123 basepair DNA ladder will be used as molecular weight markers .

II » Immunoassay

Once antibodies have been raised against the ebaf isoforms, immunoassays can be used to determine whether a bodily sample exhibits increased levels of expression of the ebaf gene . Either polyclonal or monoclonal antibodies can be used in these assays. The level of expression of the eJbaf gene observed from any of these assays should be compared with the basal level of expression the ebaf believed to be in present in healthy samples, for example, but not limited to, normal and abnormal bodily fluids including peritoneal, pericardial, pleura, senovial and cerebrospinal fluid, urine, saliva, seminal plasma, ovarian follicular fluid, peritoneal fluid, milk, etc. If the level of expression is increased relative to this basal level, it is indicative to an adenocarcinoma of the testis, or a mucinous adenocarcinoma of the colon or ovaries .

A. Western Blotting

Initially, proteins in the bodily sample are solubilized by adding to the bodily sample an equal volume of 2x SDS lysis buffer (6% SDS, 0.14 M Tris, pH 6.8, 22.4% glycerol) and the chromosomal DNA would be sheared by repeatedly passing the sample through a 20-gauge needle and then through a 26-gauge needle. The sample would then be spun at 10,000 xg for 10 minutes and the amount of protein in the supernatants would be determined using the BCA assay kit (Pierce, Rockford, 111) . Then, mercaptoethanol (5%) and bromophenol blue (0.5%) would be added and the sample will be boiled for 5 minutes . Tissue lysates would then be subjected to SDS-PAGE and separated proteins would be transferred to a nitrocellulose or nylon membrane. The membrane would be preblocked by incubation in TBST (10 mM Tris, pH 8.0; 150 mM NaCl; 0.05% Tween-20) containing 3% bovine serum albumin (BSA) at 25° C for 2 hours. After washings in TBST

(x4) , the membrane would be stained by avidin-biotin- peroxidase complex (ABC) procedure (Hsu et al, 1981) . This would be done by sequential incubation of the blot, with

TBST containing 1% BSA and primary antibody (2-12 hours) , and then with secondary antibody (2 hours) , and finally ABC (2 hours) . Each incubation would be carried at 37° C and will be followed by two washes in TBST. The immunoreactive band(s) would be revealed by incubation of the blot with a mixture of 3,3' diaminobenzidine tetrahydrochloride (DAB)-H₂0₂. As controls, primary antibody, secondary antibody or ABC would be omitted from the staining reaction. Primary antibody would be substituted with isotype specific antibody or pre-immune serum at the same protein concentration.

B. Immunohistochemical Staining

Frozen sections will be fixed in 10% buffered formalin for 5 minutes and then washed in 0.1 M PBS. If paraffin sections are used, these will be deparaffinized in xylene and descending series of ethyl alcohol and finally washed in 0.1 M PBS.

Immunostaining would be performed according to the ABC procedure as described in the Western Blot . When paraffin sections are used, if no signal can be detected, sections would be treated prior to immunostaining with pepsin or trypsin as described (Shah et al, 1987a,b) . Sections to be viewed at the light microscopic level will be evaluated with and without counterstain.

C. Enzyme Linked Immunosorbent Assay (ELISA)

ELISA is based on antigen-antibody reaction and a subsequent enzyme-mediated color development. The ELISA plates would be made in this laboratory as described in Harlow, E. and Lane, D. Antibodies : A Laboratory Manual . Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988) . If only a monoclonal antibody is available, the antibody capture assay should be used. When both polyclonal and monoclonal antibodies are available the sandwich ELISA for detecting and quantifying the antigen should be used. Initially, polyclonal antiserum would be raised in rabbits immunized with a bacteriall^ produced chimeric fusion protein or a synthesized peptide of at least one of the isoforms of produced from ebaf gene expression. If required, a second peptide would be used to raise a second polyclonal antiserum or monoclonal antibody specific to a different protein domain. Monoclonal antibodies would be synthesized by a commercial vendor (bioWorld, Dublin, OH) . The positive clones would be identified by ELISA and then expanded. The specificity of the monoclonal antibodies would be tested against the in vi tro expressed proteins.

In the antibody capture assay, known amounts of purified antigen and the bodily sample with an unknown amount of antigen would be bound to the individual wells of a PVC microtitre plate . PVC will bind approximately 100 ng/well (300 ng/cm2) . The plate would be incubated at room temperature for 2 hours . The plate would then be washed in PBS and remaining sites on the PVC plate would be saturated overnight in a humid atmosphere at room temperature with a blocking buffer (3% BSA/PBS) containing 0.2% sodium azide. After washing the plate twice with PBS, 50 ml of alkaline phosphatase-labeled antibody solution prepared in the same buffer would be added to each well and incubated for 2 hours at room temperature in a humid atmosphere. The unbound antibody would be removed by washing the plates four times in PBS.

To ensure that the assay is accurate, the amount of alkaline phosphatase-labeled antibody will be used in excess . The level of secondary antibody needed will be determined by titrating the alkaline phosphatase-labeled antibody. Once standards are prepared for the detection of known quantities of purified protein, the amount of protein in the samples will be determined using the kit. The amount of unknown protein will be extrapolated from a standard curve based on the known amounts of protein. In the sandwich ELISA, the plates would be coated with the primary monoclonal antibody (20 mg/ml in PBS) . After washing the wells with PBS, 50 ml of known amounts of purified antigen and various dilutions of lysate from the bodily sample would be added to the various wells of the plate. After washing, the alkaline phosphatase-labeled antibody to the antigen would be added and the plates re- washed. Each incubation would be for 2 hours at room temperature. A chemiluminescent detection system (Tropix, Bedfor, Massachusetts) would.be used for detection. This system includes incubation of the plates with an antibody, followed by activation of a substrate (CSPD) that emits light. The amount of light emitted would be directly related to the level of expression of the ebaf gene in the sample lysate, and that level would be quantitated by luminometry.

Applicants believe that, any method, either known now or subsequently discovered, which is capable of determining whether the ebaf gene is being expressed in a particular bodily tissue, is an acceptable method to practice the present invention. Hence, many other variations and modifications of the invention will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The above- described embodiments are, therefore, intended to be merely exemplary, and all such variations and modifications are intended to be included within the scope of the invention.

Example 3 %

Several monoclonal and polyclonal antibodies have been raised to human ebaf peptides. The polyclonal antibodies were affinity purified using the immunogenic peptides. The results of characterization of the antibodies are listed in the Table 1. The antibodies have been tested on the E-coli recombinant ebaf protein, on cells transfected with ebaf cDNA and on human endometrium and endometrial fluid (Figures 5-6) . The proteins derived from endometria were subjected to Western blot analysis using the polyclonal C (Tabibzadeh et al , 1998) and N- terminal antibodies. Both antibodies reacted with a 41 kD protein which is the predicted size of precursor protein in endometrium, endometrial fluid and placenta (Figure 5, Tabibzadeh et al, 1998) . This immunoreactivity could be inhibited by addition of excess amount of peptide (Figure 5A) . C-terminal antibody reacted with ~26, and ~32 kD proteins in endometrium (Figure 5A) . When endometrial proteins were immunoprecipitated, in addition to these proteins, an ~11 kD protein was also detected (Figure 5B) . Using this antibody, ebaf was detected in bodily samples throughout et al , 1998) . To verify the size of ebaf protein, an NIH 3T3 cell line was stably transfected with sense and antisense cDNAs of ebaf and then cell lysates and their conditioned media were subjected to Western blotting. The blot was probed with the affinity purified rabbit C-terminal antiserum to ebaf and the monoclonal antibody to C-terminal of ebaf. There was no reactivity with the cell lysates and conditioned media of cells transfected with the antisense cDNA whereas the sense cDNA induced the expression of the ebaf precursor in the cells (Figure 6) . The conditioned media of cells transfected with sense cDNA also contained the precursor protein as well as expected mature secreted ebaf proteins in the size of "11, ~26, and ~32 kD. These findings confirm that ebaf exists as a precursor protein in the size of "41 kD and secreted proteins in the size of "11, "26, and "32 kD (Table 1) .

For example, using the antibody to ebaf, the relative abundance of ebaf was examined in the endometria of infertile patients . The endometria were obtained during the receptive phase of the cycle both from infertile patients and normal controls (Figure 7) . With the exception of a 55/60 kD protein, the precursor or secreted forms of ebaf were undetectable in the endometria of fertile control (Figure 7) . On the other hand, these proteins were abundant in the endometria of infertile patients suggesting of existence of a dysregulated expression of ebaf in these endometria (Figure 7) .

In order to study ebaf by western blotting, immuno- precipitation, immunohistochemical staining and affinity purification, and to verify the specificity of the antibodies to ebaf, a number of ebaf clones with specific tags were generated by PCR (HA, Myc and FLAG) (Figure 8) . The sequence of the clones with tags was verified by an automated sequencer (Figure 8, middle panel) . 293T cells were transfected with these clones and the cell lysates and culture supernatants, were subjected to Western blot analysis.

Throughout this application, various publications, including United States patents, are referenced by author and year and patents by number. Full citations for the publications cited are listed below. The disclosures of these publications and patents in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation. Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

Table 1. Antibodies to ebaf and their c aracterizati©!π_

Antibody Atnino Acid Sequence of the immunogen ELISA Western blot analysis Western blot Western blot analysis type Tested of £ coli recombinant analysis of ebaf- of ebaf in human 26 kDa ebaf protein transfected cells endometrium

Monoclonal (N terminal) Acetyl SO clones Not done Not done Not done

C RQLQLSEVPVLDRAD (SEQ ID No.: 5) Polyclonal (N terminal) Acetyl DRADMEK VIPAC Yes Yes Not done Yes. positive

(SEQ ID No.: 6) Monoclonal (C terminal) Acetyl CASDGALVPRRLQHRP 23 clones Positive clones: 5 Positive clone: 1 Not done

(SEQ ID No.:4) Polyclonal (C teπninal) Acetyl CASDGALVPRRLQHRP Yes Positive Positive Yes. Positive (SEQ ID No.: 4)

Claims

What is Claimed is :

1. A vector which comprises the isolated nucleic acid molecule encoding endometrial bleeding associated factor having the nucleic acid sequence shown in SEQ ID No. 1.

2. A plasmid of claim 1.

3. A host vector system which comprises the vector of claim 1 and a suitable host cell for the production of a polypeptide, wherein the polypeptide is endometrial bleeding associated factor.

4. A host vector system of claim 3, wherein the suitable host cell is a bacterial cell, insect cell, or mammalian cell .

5. A method of producing a polypeptide, wherein the polypeptide is endometrial bleeding associated factor, which comprises growing the host cells of claim 3 under suitable conditions permitting production of the polypeptide and recovering the polypeptide so produced.

6. A mammalian cell comprising the vector of claim

7. A method of diagnosing an adenocarcinoma comprising the steps of : removing a bodily samples and screening the bodily sample for elevated expression of the ebaf gene.

8. The method according to claim 7 , wherein said bodily samples are bodily fluids.

9. The method according to claim 8, wherein said bodily fluids are one from the group consisting essentially of blood, urine and saliva.

10. A diagnostic tool for determining the presence of an adenocarcinoma comprising: screening means for screening a sample for the presence of ebaf protein.

11. The diagnostic tool according to claim 10, wherein said screening means is a Northern blot analysis .

12. The diagnostic tool according to claim 10, wherein said screening means is a Western blot analysis.

13. The diagnostic tool according to claim 10, wherein said screening means is an immunohistochemical stain.

14. The diagnostic tool according to claim 10, wherein said sample is a bodily fluid.

15. The diagnostic tool according to claim 14, wherein said bodily fluid is one from the group consisting essentially of blood, urine, and saliva.

16. The diagnostic tool according to claim 10, wherein the ebaf protein is detected.

17. The diagnostic tool according to claim 10, wherein the mRNA encoding ebaf is detected.

18. The diagnostic tool according to claim 10, wherein said screening means comprises an antibody.

19. The diagnostic tool according to claim 10, wherein said screening means is an immunoassay.

20. A method of diagnosing an adenocarcinoma comprising the steps of: removing a bodily sample; and screening the bodily sample for elevated levels of mRNA transcripts encoding for ebaf .

21. An isolated nucleic acid molecule encoding an endometrial bleeding associated factor (ebaf) having the nucleic acid sequence shown in SEQ. ID NO. 1.

22. An isolated DNA molecule of claim 21.

23. An isolated DNA molecule of claim 22 operatively linked to a promoter of RNA transcription.

24. An isolated cDNA molecule of claim 21.

25. An isolated cDNA molecule of claim 24 operatively linked to a promoter of RNA transcription.

26. An isolated RNA molecule of claim 21.

27. An assay for the detection of the endometrial bleeding associated factor nucleic acid molecule of claim 21.

28. A protein encoded by the nucleic acid molecule having the sequence shown in SEQ. ID NO. 1.

29. An isolated endometrial bleeding associated factor protein molecule having the amino acid sequence shown in SEQ . ID No . 3.

30. A isolated nucleic acid molecule that encodes the protein having the sequence shown in SEQ. ID. NO. 3.

31. A 1.5 kb mRNA transcript encoding an isoform of ebaf.

32. A 2.1 kb mRNA transcript encoding an isoform of ebaf.

33. A 2.5 kb mRNA transcript encoding an isoform of ebaf.

34. An antibody directed to ebaf .

35. An antibody directed to a peptide having the amino acid sequence shown in SEQ ID No : .

36. An antibody directed to a peptide having the amino acid sequence shown in SEQ ID No : 5.

37. An antibody directed to a peptide having the amino acid sequence shown in SEQ ID No: 6.

38. A peptide epitope of ebaf having the amino acid sequence shown in SEQ . ID NO . 4.

39. A peptide epitope of ebaf having the amino acid sequence shown in SEQ . ID NO . 5.

40. A peptide epitope of ebaf having the amino acid sequence shown in SEQ . ID NO . 6.

41. An antibody directed to a peptide epitope of ebaf.

42. An assay for determining the presence of ebaf .