Patent application title: METHOD FOR DIAGNOSING SELECTED ADENOCARCINOMAS
Siamak Tabibzadeh (Albertson, NY, US)
Ravi Kothapalli (Wesley Chapel, FL, US)
University of South Florida
IPC8 Class: AC07H2104FI
Class name: Nitrogen containing n-glycosides, polymers thereof, metal derivatives (e.g., nucleic acids, oligonucleotides, etc.) dna or rna fragments or modified forms thereof (e.g., genes, etc.)
Publication date: 2010-11-18
Patent application number: 20100292451
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.
1. An isolated polynucleotide that hybridizes to the ebaf gene.
2. An isolated polynucleotide, wherein the isolated polynucleotide hybridizes to the polynucleotide of SEQ ID NO: 1.
CROSS-REFERENCE TO RELATED APPLICATION
The subject application is a divisional of U.S. patent application Ser. No. 11/654,074, filed Jan. 17, 2007, which is a continuation of U.S. patent application Ser. No. 10/206,508, filed Jul. 26, 2002, now abandoned, which is a divisional of U.S. patent application Ser. No. 09/526,833, filed Mar. 16, 2000, now U.S. Pat. No. 6,683,156, which is a divisional of U.S. patent application Ser. No. 09/342,819, filed Jun. 29, 1999, now U.S. Pat. No. 6,294,662, which is a continuation of U.S. patent application Ser. No. 08/919,421, filed Aug. 27, 1997, now U.S. Pat. No. 5,916,751, which claims priority to U.S. Provisional Patent Application Ser. No. 60/025,800, filed Aug. 27, 1996, the disclosure of each of which is incorporated herein by reference in its entirety, including all figures, tables, amino acid and nucleic acid sequences.
FIELD OF THE 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 THE 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 hone 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. Hence, this 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 FORT result.
However, 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 non-inflammatory 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 patients 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. Hence 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.
However, 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., et al. An Evaluation of the Carcinoembryonic Antigen (CEA) Test for monitoring Patients with resected Colon Cancer. JAMA 270(8):354 (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 respectable 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 Adults JAMA (1989) 261(4):59S)
However, 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 25,000 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. However, like 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 TEE INVENTION
There is provided, in accordance with the present invention, 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 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, or a sample of blood. 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 (please see Ravi Kothapalli, Ibrahim Buyuksal, Shi-Qi Wu, Nasser Chegini, Siamak Tabibzadeh: Detection of ebaf, a novel human gene of the TGF-? superfamily; association of gene expression with endometrial bleeding J. Olin. 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 or the present invention is to provide an accurate, reliable method for the diagnosis and detection of a mutinous 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 (SCC), 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 blood test for adenocarcinomas of the testis, and mutinous adenocarcinomas of the colon and ovaries. As stated above, only 6 organs are presently known to express the ebaf cane constitutively. Applicants believe this constitutive expression results in a basal level of expression of the ebaf gene in the blood. However, if increased levels of expression of the ebaf gene are detected in the blood of a human relative to the basal level, they indicate the presence of an adenocarcinoma of the testis, or a mutinous adenocarcinoma of the talon or ovary. For example, if increased levels of expression of the ebaf gene are detected in a blood 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 blood 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
FIG. 1 is a Northern blot analysis of ebaf mRNA in normal tissues;
FIG. 2 is a Northern blot analysis of ebaf mRNA in colonic adenocarcinomas; and
FIG. 3 is a Northern blot analysis of ebaf mRNA in testicular cancers.
DESCRIPTION OF EXAMPLES OF TEE INVENTION
In the description of examples of the invention, applicants principally, used the Northern Blot method to determine the expression of the ebaf gene in a particular bodily samples made up of tissues from different organs. 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 ebaf 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.
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×g 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 isopropanol 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, Calif.). The RNA was fixed to the membrane by UV cross-linking. Using a "PRIME-A-GENE" kit, cDNA was labeled with [32P] to a high specific activity, and purified by Nick columns. Membranes were prehybridized in 50% formamide, 10×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 106 cpm/ml of heat-denatured probe in the same buffer containing 10% dextran sulfate. Then, membranes were sequentially washed three times in 4×SSC, one time in 0.5×SSC and then one time in 0.1×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, 0.1× saline sodium phosphate ETDA (SSPE), and 0.2% SDS at 50° C. for one hour.
FIG. 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 (lane 2: spleen, lane 2: 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 ebaf cDNA (SEQ. NO. 1) as the probe. As shown, a band of ebaf 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 ebaf 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-US-00001 TABLE 1 Expression of ebaf mRNA in Normal Tissues Number of Northern blot Tissue tissues examined finding Breast 5 -- Stomach 3 -- Small Bowel 1 -- Colon* 11 2.1 kb (weak) Rectum 2 2.1 kb Liver 6 -- Pancreas 2 2.1 and 2.5 kb Kidney 1 2.5 (weak) Lung 1 -- Fallopian Tube 1 -- Ovary 7 -- Ovary 1 2.1 kb Testis 2 2.1 kb Spleen 1 -- Lymph node 1 -- --: signal not detected *of eleven samples tested, only one showed a strong signal. The remaining were not apparent.
The expression of the ebaf 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 froth 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 ebaf 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-US-00002 TABLE 2 Expression of ebaf mRNA in the Non-Mucinous Adenocarcinomas Number of tumors Northern Tumor Type examined blot finding Serous Cystadenocarcinoma of ovary 2 2.1 Serous Cystadenocarcinoma of ovary 2 2.5 Serous Cystadenocarcinoma of ovary 5 -- Adenocarcinoma of the colon metastatic to 1 -- ovary Endometrioid adenocarcinoma of ovary 1 -- Non-mucinous adenocarcinoma of colon 7 -- Non-mucinous adenocarcinoma of uterine 1 -- cervix Non-mucinous adenocarcinoma of the 3 -- stomach Hepatocellular carcinoma 3 -- Renal Cell Carcinoma 3 -- Liver metastasis; Consistent with colonic 6 -- primary* Adenocarcinoma of lung 7 -- Adenocarcinoma of breast 5 -- *Normal tissues around the tumors were available for the Northern blot analysis and did not exhibit ebaf 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-US-00003 TABLE 3 Expression of ebaf mRNA in Squamous Call Carcinomas Number of Northern tumors blot Tumor Type examined finding SCC of the Larynx 1 -- SCC of the Lung 4 -- SCC of the Uterine cervix 1 -- --: Signal not detected
TABLE-US-00004 TABLE 4 Expression of ebaf mRNA in Non-epithelial Tumors Number of Northern tumors blot Tumor Type examined finding Leiomyosarcoma, gastric 1 -- Leiomyosarcoma, colon 1 -- Leiomyosarcoma, pelvic 1 -- Chondrosarcoma, thoracic 1 -- wall Osteosarcoma, metastatic to 3 -- the lung Liposarcoma, 1 -- retroperitoneum Synovial sarcoma, metastatic 1 -- to the chest wall Synovial sarcoma, parotid 1 -- Synovial sarcoma, Leg -- Angiosarcoma, mediastinal 1 -- Lymphoma 1 -- Lymphoma, B cell type 1 -- Lymphoma, B cell, spleen 1 -- Lymphoma, T cell, groin 1 -- Lymphoma, T cell, 1 -- angiocentric, hip Hodgkin's Disease, mixed 1 -- cell type, lymph node Melanoma 5 -- --: signal not detected
Detection of Mucinous Adenocarcinomas of the Colon
In FIG. 2, 20 mg total RNA from a normal late secretory endometrium which served as the positive control M (lane 1) as well as mucinous adenocarcinomas of colon (lanes 2, 4, 6), non-mucinous adenocarcinomas of colon (lanes 9, 10, and 12) and adjacent normal colon (lanes 3, 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.
Detection of Adenocarcinomas of the Testis
FIG. 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 ebaf 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 FIG. 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).
II. 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, Ohio) 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 KCl, 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, Calif.), 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.
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 ebaf 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. 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 2×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×g for 10 minutes and the amount of protein in the supernatants would be determined using the BCA assay kit (Pierce, Rockford, Ill.). 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 two hours. After washings in TEST (×4), the membrane would be stained by avidin-biotin-peroxidase complex (ABC) procedure (Hsu at al, 1981). This would be done by sequential incubation of the blot, with TEST 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 TEST. The immunoreactive band(s) would be revealed by incubation of the blot with a mixture of 3,3' diaminobenzidine tetrahydrochloride (DAB)-H2O2. 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 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, N.Y. (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 bacterially 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, Ohio). The positive clones would be identified by ELISA and then expanded. The specificity of the monoclonal antibodies would be tested against the in vitro 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 two 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 two 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 two hours at room temperature. A chemiluminescent detection system (Tropix, Bedfor, Mass.) 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.
311962DNAHomo sapiens 1ccccactctg cctcctgctc ccccagggca gcaccatgtg gcccctgtgg ctctgctggg 60cactctgggt gctgcccctg gctggccccg gggcggccct gaccgaggag cagctcctgg 120cgagcctgct gcggcagctg cagctcagcg aggtgcccgt actggacagg gccgacatgg 180agaagctggt catccccgcc cacgtgaggg cccagtatgt agtcctgctg cggcgcgacg 240gggaccgctc ccgcggaaag aggttcagcc agagcttccg agaggtggcc ggcaggttcc 300tggcgtcgga ggccagcaca cacctgctgg tgttcggcat ggagcagcgg ctgccgccca 360acagcgagct ggtgcaggcc gtgctgcggc tcttccagga gccggttccc caaggcgcgc 420tgcacaggca cgggcggctg tccccggcag cgcccaaggc ccgggtgacc gtcgagtggc 480tggtccgcga cgacggctcc aaccgcacct ccctcatcga ctccaggctg gtgtccgtcc 540acgagagcgg ctggaaggcc ttcgacgtga ccgaggccgt gaacttctgg cagcagctga 600gccggccccc ggagccgctg ctcgtacagg tgtcggtgca gagggagcat ctgggcccgc 660tggcgtccgg cgcccacaag ctggtccgct ttgcctcgca gggggcgcca gccgggcttg 720gggagcccca gctggagctg cacaccctgg acctcaggga ctatggagct cagggcgact 780gtgaccctga agcaccaatg accgagggca cccgctgctg ccgccaggag atgtacattg 840acctgcaggg gatgaagtgg gccaagaact gggtgctgga gcccccgggc ttcctggctt 900acgagtgtgt gggcacctgc cagcagcccc cggaggccct ggccttcaat tggccatttc 960tggggccgcg acagtgtatc gcctcggaga ctgcctcgct gcccatgatc gtcagcatca 1020aggagggagg caggaccagg ccccaggtgg tcagcctgcc caacatgagg gtgcagaagt 1080gcagctgtgc ctcggatggg gcgctcgtgc caaggaggct ccagcatagg ccctggtgta 1140tccattgagc ctctaactga acgtgtgcat aagaggtggt cttaatgtag ggcgttaact 1200ttatacttag caagttactc catcccaatt tagtgctcct gtgtgacctc gccctgtgtc 1260cttccattcc tgtctttccc gtccatcacc catcctaagc acttacgtga gtaaataatg 1320cagctcagat gctgagctct agtaggaaat gctggcatgc tgattacaag atacagctga 1380gcaatgcaca cattttcagc tgggagtttc tgttctctgg caaattcttc actgagtctg 1440gaacaataat accctatgat tagaactggg gaaacagaac tgaattgctg tgttatatga 1500ggaattaaaa ccttcaaatc tctatttccc ccaaatactg acccattctg gacttttgta 1560aacataccta ggcccctgtt cccctgagag ggtgctaaga ggaaggatga gggcttcagg 1620ctgggggcag tggacaggga attgggatac ctggattctg gttctgacag ggccacaagc 1680taggatctct aacaaacgca gaaggctttg gctcgtcatt tcctcttaaa aaaggaggag 1740ctgggcttca gctctaagaa cttcattgcc ctggggatca gacagcccct acctacccct 1800gcccactcct ctggagactg agccttgccc gtgcatattt aggtcatttc ccacactgtc 1860ttagagaact tgtcaccaga aaccacatgt atttgcatgt tttttgttaa tttagctaaa 1920gcaattgaat gtagatactc agaagaaata aaaaatgatg tt 196221961DNAHomo sapiensmisc_feature(1)..(1961)Nucleotide and deduced amino acid sequences of ebaf. 2ccactctgcc tcctgctccc ccagggcagc acc atg tgg ccc ctg tgg ctc tgc 54 Met Trp Pro Leu Trp Leu Cys 1 5tgg gca ctc tgg gtg ctg ccc ctg gct ggc ccc ggg gcg gcc ctg acc 102Trp Ala Leu Trp Val Leu Pro Leu Ala Gly Pro Gly Ala Ala Leu Thr 10 15 20gag gag cag ctc ctg gcg agc ctg ctg cgg cag ctg cag ctc agc gag 150Glu Glu Gln Leu Leu Ala Ser Leu Leu Arg Gln Leu Gln Leu Ser Glu 25 30 35gtg ccc gta ctg gac agg gcc gac atg gag aag ctg gtc atc ccc gcc 198Val Pro Val Leu Asp Arg Ala Asp Met Glu Lys Leu Val Ile Pro Ala40 45 50 55cac gtg agg gcc cag tat gta gtc ctg ctg cgg cgc gac ggg gac cgc 246His Val Arg Ala Gln Tyr Val Val Leu Leu Arg Arg Asp Gly Asp Arg 60 65 70tcc cgc gga aag agg ttc agc cag agc ttc cga gag gtg gcc ggc agg 294Ser Arg Gly Lys Arg Phe Ser Gln Ser Phe Arg Glu Val Ala Gly Arg 75 80 85ttc ctg gcg tcg gag gcc agc aca cac ctg ctg gtg ttc ggc atg gag 342Phe Leu Ala Ser Glu Ala Ser Thr His Leu Leu Val Phe Gly Met Glu 90 95 100cag cgg ctg ccg ccc aac agc gag ctg gtg cag gcc gtg ctg cgg ctc 390Gln Arg Leu Pro Pro Asn Ser Glu Leu Val Gln Ala Val Leu Arg Leu 105 110 115ttc cag gag ccg gtt ccc caa ggc gcg ctg cac agg cac ggg cgg ctg 438Phe Gln Glu Pro Val Pro Gln Gly Ala Leu His Arg His Gly Arg Leu120 125 130 135tcc ccg gca gcg ccc aag gcc cgg gtg acc gtc gag tgg ctg gtc cgc 486Ser Pro Ala Ala Pro Lys Ala Arg Val Thr Val Glu Trp Leu Val Arg 140 145 150gac gac ggc tcc aac cgc acc tcc ctc atc gac tcc agg ctg gtg tcc 534Asp Asp Gly Ser Asn Arg Thr Ser Leu Ile Asp Ser Arg Leu Val Ser 155 160 165gtc cac gag agc ggc tgg aag gcc ttc gac gtg acc gag gcc gtg aac 582Val His Glu Ser Gly Trp Lys Ala Phe Asp Val Thr Glu Ala Val Asn 170 175 180ttc tgg cag cag ctg agc cgg ccc ccg gag ccg ctg ctc gta cag gtg 630Phe Trp Gln Gln Leu Ser Arg Pro Pro Glu Pro Leu Leu Val Gln Val 185 190 195tcg gtg cag agg gag cat ctg ggc ccg ctg gcg tcc ggc gcc cac aag 678Ser Val Gln Arg Glu His Leu Gly Pro Leu Ala Ser Gly Ala His Lys200 205 210 215ctg gtc cgc ttt gcc tcg cag ggg gcg cca gcc ggg ctt ggg gag ccc 726Leu Val Arg Phe Ala Ser Gln Gly Ala Pro Ala Gly Leu Gly Glu Pro 220 225 230cag ctg gag ctg cac acc ctg gac ctc agg gac tat gga gct cag ggc 774Gln Leu Glu Leu His Thr Leu Asp Leu Arg Asp Tyr Gly Ala Gln Gly 235 240 245gac tgt gac cct gaa gca cca atg acc gag ggc acc cgc tgc tgc cgc 822Asp Cys Asp Pro Glu Ala Pro Met Thr Glu Gly Thr Arg Cys Cys Arg 250 255 260cag gag atg tac att gac ctg cag ggg atg aag tgg gcc aag aac tgg 870Gln Glu Met Tyr Ile Asp Leu Gln Gly Met Lys Trp Ala Lys Asn Trp 265 270 275gtg ctg gag ccc ccg ggc ttc ctg gct tac gag tgt gtg ggc acc tgc 918Val Leu Glu Pro Pro Gly Phe Leu Ala Tyr Glu Cys Val Gly Thr Cys280 285 290 295cag cag ccc ccg gaa gcc ctg gcc ttc aat tgg cca ttt ctg ggg ccg 966Gln Gln Pro Pro Glu Ala Leu Ala Phe Asn Trp Pro Phe Leu Gly Pro 300 305 310cga cag tgt atc gcc tcg gag act gcc tcg ctg ccc atg atc gtc agc 1014Arg Gln Cys Ile Ala Ser Glu Thr Ala Ser Leu Pro Met Ile Val Ser 315 320 325atc aag gag gga ggc agg acc agg ccc cag gtg gtc agc ctg ccc aac 1062Ile Lys Glu Gly Gly Arg Thr Arg Pro Gln Val Val Ser Leu Pro Asn 330 335 340atg agg gtg cag aag tgc agc tgt gcc tcg gat ggg gcg ctc gtg cca 1110Met Arg Val Gln Lys Cys Ser Cys Ala Ser Asp Gly Ala Leu Val Pro 345 350 355agg agg ctc cag cat agg ccc tgg tgt atc cat tgagcctcta actgaacgtg 1163Arg Arg Leu Gln His Arg Pro Trp Cys Ile His360 365 370tgcataagag gtggtcttaa tgtagggcgt taactttata cttagcaagt tactccatcc 1223caatttagtg ctcctgtgtg acctcgccct gtgtccttcc attcctgtct ttcccgtcca 1283tcacccatcc taagcactta cgtgagtaaa taatgcagct cagatgctga gctctagtag 1343gaaatgctgg catgctgatt acaagataca gctgagcaat gcacacattt tcagctggga 1403gtttctgttc tctggcaaat tcttcactga gtctggaaca ataataccct atgattagaa 1463ctggggaaac agaactgaat tgctgtgtta tatgaggaat taaaaccttc aaatctctat 1523ttcccccaaa tactgaccca ttctggactt ttgtaaacat acctaggccc ctgttcccct 1583gagagggtgc taagaggaag gatgaagggc ttcaggctgg gggcagtgga cagggaattg 1643ggatacctgg attctggttc tgacagggcc acaagctagg atctctaaca aacgcagaag 1703gctttggctc gtcatttcct cttaaaaaag gaggagctgg gcttcagctc taagaacttc 1763attgccctgg ggatcagaca gcccctacct acccctgccc actcctctgg agactgagcc 1823ttgcccgtgc atatttaggt catttcccac actgtcttag agaacttgtc accagaaacc 1883acatgtattt gcatgttttt tgttaattta gctaaagcaa ttgaatgtag atactcagaa 1943gaaataaaaa atgatgtt 19613370PRTHomo sapiens 3Met Trp Pro Leu Trp Leu Cys Trp Ala Leu Trp Val Leu Pro Leu Ala1 5 10 15Gly Pro Gly Ala Ala Leu Thr Glu Glu Gln Leu Leu Ala Ser Leu Leu 20 25 30Arg Gln Leu Gln Leu Ser Glu Val Pro Val Leu Asp Arg Ala Asp Met 35 40 45Glu Lys Leu Val Ile Pro Ala His Val Arg Ala Gln Tyr Val Val Leu 50 55 60Leu Arg Arg Asp Gly Asp Arg Ser Arg Gly Lys Arg Phe Ser Gln Ser65 70 75 80Phe Arg Glu Val Ala Gly Arg Phe Leu Ala Ser Glu Ala Ser Thr His 85 90 95Leu Leu Val Phe Gly Met Glu Gln Arg Leu Pro Pro Asn Ser Glu Leu 100 105 110Val Gln Ala Val Leu Arg Leu Phe Gln Glu Pro Val Pro Gln Gly Ala 115 120 125Leu His Arg His Gly Arg Leu Ser Pro Ala Ala Pro Lys Ala Arg Val 130 135 140Thr Val Glu Trp Leu Val Arg Asp Asp Gly Ser Asn Arg Thr Ser Leu145 150 155 160Ile Asp Ser Arg Leu Val Ser Val His Glu Ser Gly Trp Lys Ala Phe 165 170 175Asp Val Thr Glu Ala Val Asn Phe Trp Gln Gln Leu Ser Arg Pro Pro 180 185 190Glu Pro Leu Leu Val Gln Val Ser Val Gln Arg Glu His Leu Gly Pro 195 200 205Leu Ala Ser Gly Ala His Lys Leu Val Arg Phe Ala Ser Gln Gly Ala 210 215 220Pro Ala Gly Leu Gly Glu Pro Gln Leu Glu Leu His Thr Leu Asp Leu225 230 235 240Arg Asp Tyr Gly Ala Gln Gly Asp Cys Asp Pro Glu Ala Pro Met Thr 245 250 255Glu Gly Thr Arg Cys Cys Arg Gln Glu Met Tyr Ile Asp Leu Gln Gly 260 265 270Met Lys Trp Ala Lys Asn Trp Val Leu Glu Pro Pro Gly Phe Leu Ala 275 280 285Tyr Glu Cys Val Gly Thr Cys Gln Gln Pro Pro Glu Ala Leu Ala Phe 290 295 300Asn Trp Pro Phe Leu Gly Pro Arg Gln Cys Ile Ala Ser Glu Thr Ala305 310 315 320Ser Leu Pro Met Ile Val Ser Ile Lys Glu Gly Gly Arg Thr Arg Pro 325 330 335Gln Val Val Ser Leu Pro Asn Met Arg Val Gln Lys Cys Ser Cys Ala 340 345 350Ser Asp Gly Ala Leu Val Pro Arg Arg Leu Gln His Arg Pro Trp Cys 355 360 365Ile His 370
Patent applications by Ravi Kothapalli, Wesley Chapel, FL US
Patent applications by Siamak Tabibzadeh, Albertson, NY US
Patent applications by University of South Florida
Patent applications in class DNA or RNA fragments or modified forms thereof (e.g., genes, etc.)
Patent applications in all subclasses DNA or RNA fragments or modified forms thereof (e.g., genes, etc.)