EP0538430A1 - Endometrial antigen, composition, test kit and method for endometrial antibody determination - Google Patents
Endometrial antigen, composition, test kit and method for endometrial antibody determinationInfo
- Publication number
- EP0538430A1 EP0538430A1 EP92909247A EP92909247A EP0538430A1 EP 0538430 A1 EP0538430 A1 EP 0538430A1 EP 92909247 A EP92909247 A EP 92909247A EP 92909247 A EP92909247 A EP 92909247A EP 0538430 A1 EP0538430 A1 EP 0538430A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- fragment
- kilodaltons
- molecular weight
- endometrial
- antigen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57442—Specifically defined cancers of the uterus and endometrial
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
- G01N2800/364—Endometriosis, i.e. non-malignant disorder in which functioning endometrial tissue is present outside the uterine cavity
Definitions
- endometrial antigens having various molecular weights were described as isolated from cultures or culture media obtained from several epithelial carcinoma cell lines. Monoclonal antibodies and immunological reagents directed to the antigens are also described. The antibodies and antigens were then used to detect endometrial antibodies in patient specimens using various immunological procedures. It would be desirable to have additional antigens which could be used in a sensitive and accurate assay for endometrial antibodies.
- the isolated antigen or mixture thereof can be supplied in a buffered composition for use in various immunological methods.
- the composition is generally buffered to a pH of from about 6 to about 8 using one or more suitable buffers such as phosphate buffered saline solution, tris(hydroxymethyl)amino ⁇ methane, glycine, 3-(N-morpholino)propanesulfonic acid, borates, and others known in the art, [for example, Good et al, Biochem.. ⁇ ., 467 (1966)], most of which are commercially available.
- the amount of antigen in such a composition can vary widely depending upon its intended use.
- the isolated antigen fragments can be used in crude form (that is, in admixture with extraneous cellular materials) or at various levels of purification.
- Useful enzyme labels include peroxidase, alkaline phosphatase, urease, glucose oxidase, beta- ⁇ alactosidase and others readily apparent to one skilled in the art. Peroxidase and alkaline phosphatase are preferred.
- the particles are generally greater than about 0.05 m meters in diameter.
- Useful materials for such supports would also be apparent to one skilled in the art particularly in view of the teaching in EP-A-0 387 027.
- the antigens can be attached to such materials by adsorption or other non- covalent means (see for example US-A-4,528,267) or covalent means using techniques generally known, including those described above for coupling particulate labels to antigen, and others described by Chibata, Immobilized Enzvmes. Halsted Press: New York (1978) and Cautrecasas, J.Bio.Chem.. 245. 1059 (1970) .
- coupling can be directly to the support through reactive groups or ionic bonds, or through coupling moieties or proteins as is known in the art.
- the antigens can be coupled to microtiter plates by non-specific adsorption or covalent coupling to a reactive group on the plate.
- Non-human monoclonal antibodies can also be prepared using the standard method of K ⁇ hler et al. Nature. 256. pp. 495-497 (1975) involving the use of hybridomas prepared from immunized mice or rats to produce suspended spleen cells. Suitable hybridomas are available from either commercial sources or various cell culture collections including the ATCC.
- antibodies including human monoclonal antibodies specific to the antigen and antiidiotypic antibodies can be prepared using the procedures described in more detail in EP-A-0 387 027.
- the antigens of this invention are useful for the detection (that is, measuring the presence, amount or both) of endometrial antibodies found in human body fluids, such as whole blood, blood serum, suspensions of endometrial tissues, peritoneal fluid and uterine fluid or secretions.
- the antibodies are detected in blood serum.
- These antibodies are generally identified as human IgG type antibodies although IgA type antibodies may also be present.
- kits may include the compositions and reagents (noted above) used in particular assays as well as the necessary instructions, test devices, specimen handling equipment for assaying one or more specimens.
- the kit includes the antigen (or mixture thereof) , labeled anti-human antibodies reactive with the endometrial antibodies, and a means for detecting the resulting immunological reaction.
- the detecting means can be a test device, microtiter plate, a dye-providing composition or others known in the art, or a combination thereof.
- the extracts were resolved using SDS-PAGE electrophoresis with a 10% uniform polyacrylamide reducing gel in a buffer solution of tris(hydroxymethyl)aminomethane buffer (25 mmolar, pH 8.5), glycine buffer (200 mmolar), sodium dodecylsulfate (0.1%) and sodium acetate (100 mmolar) for 3-4 hours, increasing the voltage to 400 volts.
- Isoelectric point (pi) was determined by two- dimensional electrophoresis.
- the cell lysate proteins, prepared as described above, were first separated by isoelectric point using isoelectric focusing in the first dimension, and then the electrofocused proteins were separated according to molecular weight by SDS- PAGE electrophoresis in the second dimension.
- the positive serum antigens (which are the 63-67 kD, 40-44 kD, 33-37 kD, 57-59 kD and 59- 64 kD antigens) were compared to the negative serum antigens (which are common to negative and positive serum: which may include 30-32 kD, 18-22 kD, 47-50 kD and 28-30 kD antigens) .
- Antigens unique to the positive serum were then noted and the molecular weights and isoelectric points calculated. In other words, the antigens were identified by subtracting the common bands on the immunoblots from the bands shown on the immunoblot for the positive serum sample.
- the isolated antigen fragments and available data are listed in Table II below.
- the molecular weight of each fragment is listed as a narrow range because the exact value is difficult to determine using known procedures and a 10% variation is generally accepted in the art.
- Example 3 Preparation of Detectably Labeled Endometrial Antibody Reagent This is a prospective example of how a detectably labeled endometrial antibody reagent of this invention can be prepared. This reagent would comprise an endometrial antigen fragment (isolated as described above) which is labeled with 5-dimethylamino- naphthalene-1-sulfonyl chloride, a fluorescent moiety.
- the reagent could be prepared by adding a 3- to 5-fold molar excess of 5-dimethylaminonaphthalene-l- sulfonyl chloride to isolated antigen fragment in tris(hydroxymethyl)aminomethane buffer (25 mmolar, pH 8.3) . Before the addition, the chloride is dissolved in acetone equal to 1% of the final volume. Reaction would proceed for 1 to 2 hours at room temperature, and the resulting mixture dialyzed in the buffer (25 mmolar, pH 8.3) to provide the desired reagent.
- Example 4 Preparation of Endometrial Antibody Capture Reagent This is an example of the preparation of an endometrial antibody capture reagent of this invention.
- E-]_ endometrial antigens
- E2 E and E5 were isolated as described in Example 1.
- the antigen fragments were further purified as follows: Extraneous proteins were removed by precipitating them at 25% ammonium sulfate and centrifugation. The pellet was discarded, and the supernatant containing the antigen fragments was treated by adding ammonium sulfate to 40% and centrifugation, and the resulting pellet was resuspended in tris(hydroxymethyl)aminomethane buffer (pH 7.5). This suspension was dialyzed against buffer.
- the resulting solution was diluted to 30 ⁇ g protein/ml in phosphate buffered saline solution containing protease inhibitors, and a sample (100 ⁇ l) was added to each well of a polystyrene microtiter plate and incubated at room temperature for two hours. The plate was then washed three times with phosphate buffered saline solution. Remaining binding sites on the plates were blocked with bovine serum albumin (3%) in phosphate buffered saline solution for two hours at room temperature. The plate was then washed three times with a solution of TWEENTM 20 nonionic surfactant (0.05%) in phosphate buffered saline solution.
- nonspecific protein binding sites were blocked for one hour at 24°C with a "blocking" solution of tris(hydroxymethyl)aminomethane (20 mmolar, pH 7.5) containing gelatin (3%), normal goat serum (0.4%) and sodium chloride (500 mmolar) .
- the blocked nitrocellulose was then washed twice, 5 minutes each time, with tris(hydroxymethyl)aminomethane (20 mmolar, pH 7.5) containing sodium chloride (500 mmolar) and
- TWEEN 20 nonionic surfactant 0.05%).
- the nitrocellulose strips and diluted serum samples (10 ml) were then contacted for incubation for two hours at 24°C.
- the serum had been diluted 100-fold in tris(hydroxymethyl)aminomethane (20 mmolar, pH 7.5) containing gelatin (1%) , sodium chloride (500 mmolar) ,
- nitrocellulose strips were washed four times (5 minutes each time) with the buffered solution containing TWEEN TM 20 noted above (30 ml) to remove uncomplexed materials.
- the nitrocellulose strips were washed four times (5 minutes each) with the buffered TWEEN 20 solution noted above, and once (5 minutes) with the buffer solution noted above without TWEEN TM 20 to remove uncomplexed reactants and excess TWEEN TM 20 nonionic surfactant.
- Tables III and IV below show the results of the sera screen using the assay noted above.
- the antigens used for obtaining the data in Table III were extracted using phosphate buffered saline solution while Table IV data were obtained using antigens extracted using the buffered sucrose solution described in Example 1 above.
- FIGURE as "42 kD” and “35 kD”, respectively). Bands lc, Id, 2c and 2d are negative controls and do not show bands for the noted fragments.
- the dye stop solution comprised sodium azide (0.1%) .
- the dye-providing composition (40 ⁇ l) was then added to each test well. The fluid was allowed to drain and the test was incubated for 2 minutes. The dye stop solution (80 ⁇ l) was then added, followed by fluid drainage.
- Serum Sample known to be positive for antibodies
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Reproductive Health (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Plusieurs fragments antigènes protéiques ont été isolés du cytoplasme de cellules épithéliales adénocarcinomiques. Les antigènes protéiques sont utiles pour détecter les anticorps endométriaux, lesquels révèlent une endométriose. Les antigènes peuvent être liés à des supports non solubles dans l'eau ou marqués de manière à pouvoir être détectés, afin de constituer des réactifs. La détection d'anticorps endométriaux s'effectue en faisant réagir l'antigène aux anticorps d'un échantillon puis en détectant le complexe résultant. Les antigènes peuvent êtres fournis sous forme de composition tamponnée dans une trousse pour épreuve diagnostique.Several protein antigen fragments have been isolated from the cytoplasm of adenocarcinomic epithelial cells. Protein antigens are useful for detecting endometrial antibodies, which reveal endometriosis. The antigens can be linked to supports that are not soluble in water or can be labeled so that they can be detected in order to constitute reagents. The detection of endometrial antibodies is carried out by reacting the antigen with the antibodies in a sample and then detecting the resulting complex. The antigens can be supplied as a buffered composition in a diagnostic test kit.
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US68217791A | 1991-04-09 | 1991-04-09 | |
US682177 | 2001-07-31 |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0538430A1 true EP0538430A1 (en) | 1993-04-28 |
Family
ID=24738559
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP92909247A Withdrawn EP0538430A1 (en) | 1991-04-09 | 1992-04-09 | Endometrial antigen, composition, test kit and method for endometrial antibody determination |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP0538430A1 (en) |
JP (1) | JPH05507095A (en) |
KR (1) | KR930700548A (en) |
CA (2) | CA2062675A1 (en) |
FI (1) | FI925593A0 (en) |
IE (1) | IE921132A1 (en) |
WO (1) | WO1992018535A1 (en) |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994028021A1 (en) * | 1993-05-28 | 1994-12-08 | Medical University Of South Carolina | Endometrial proteins, antigenic compositions and methods for detecting endometriosis |
US5618680A (en) * | 1994-12-28 | 1997-04-08 | Institut De Medecine De La Reproduction De Montreal | Use of ligands specific to major histocompatibility complex-class I antigens for diagnosing endometriosis |
US6376201B2 (en) | 1994-12-28 | 2002-04-23 | Procrea Biosciences Inc. | Use of ligands specific to major histocompatibility complex-class I antigens for diagnosing endometriosis |
US6677128B1 (en) | 1997-06-26 | 2004-01-13 | Regents Of The University Of Michigan | Method for identification of cellular protein antigens and presence of antibodies to specific cellular protein antigens in serum |
ATE266203T1 (en) * | 1997-06-26 | 2004-05-15 | Univ Michigan | METHOD FOR IDENTIFYING TUMOR ANTIGENS WITH AUTOANTIBODIES IN SERUM |
JP4820003B2 (en) * | 1998-11-05 | 2011-11-24 | ザ リージェンツ オブ ザ ユニバーシティ オブ ミシガン | S100 protein and autoantibodies as serum markers for cancer |
US6743595B1 (en) | 1999-01-25 | 2004-06-01 | Metriogene Biosciences Inc. | Method and diagnostic kit for diagnosis of endometriosis |
US20060008876A1 (en) | 2004-07-07 | 2006-01-12 | Shami A S E | ME-5, ME-2, and EPP2: human protein antigens reactive with autoantibodies present in the serum of women suffering from endometriosis |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
NZ232801A (en) * | 1989-03-07 | 1992-01-29 | Adeza Biomedical Corp | Diagnostic test for endometriosis |
-
1992
- 1992-03-11 CA CA002062675A patent/CA2062675A1/en not_active Abandoned
- 1992-04-09 IE IE113292A patent/IE921132A1/en unknown
- 1992-04-09 EP EP92909247A patent/EP0538430A1/en not_active Withdrawn
- 1992-04-09 JP JP92508868A patent/JPH05507095A/en active Pending
- 1992-04-09 CA CA002081900A patent/CA2081900A1/en not_active Abandoned
- 1992-04-09 WO PCT/US1992/002888 patent/WO1992018535A1/en not_active Application Discontinuation
- 1992-12-09 FI FI925593A patent/FI925593A0/en unknown
- 1992-12-09 KR KR1019920703159A patent/KR930700548A/en not_active Application Discontinuation
Non-Patent Citations (1)
Title |
---|
See references of WO9218535A1 * |
Also Published As
Publication number | Publication date |
---|---|
CA2081900A1 (en) | 1992-10-10 |
CA2062675A1 (en) | 1992-10-10 |
WO1992018535A1 (en) | 1992-10-29 |
IE921132A1 (en) | 1992-10-21 |
FI925593A (en) | 1992-12-09 |
FI925593A0 (en) | 1992-12-09 |
KR930700548A (en) | 1993-03-15 |
JPH05507095A (en) | 1993-10-14 |
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Legal Events
Date | Code | Title | Description |
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PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
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17P | Request for examination filed |
Effective date: 19921207 |
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17Q | First examination report despatched |
Effective date: 19950421 |
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RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: CLINICAL DIAGNOSTIC SYSTEMS, INC. |
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RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: JOHNSON & JOHNSON CLINICAL DIAGNOSTICS, INC. |
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STAA | Information on the status of an ep patent application or granted ep patent |
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Effective date: 19971104 |