WO1992018535A1 - Antigene endometrial, composition, trousse pour epreuve et methode de determination d'anticorps endometriaux - Google Patents

Antigene endometrial, composition, trousse pour epreuve et methode de determination d'anticorps endometriaux Download PDF

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Publication number
WO1992018535A1
WO1992018535A1 PCT/US1992/002888 US9202888W WO9218535A1 WO 1992018535 A1 WO1992018535 A1 WO 1992018535A1 US 9202888 W US9202888 W US 9202888W WO 9218535 A1 WO9218535 A1 WO 9218535A1
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Prior art keywords
fragment
kilodaltons
molecular weight
endometrial
antigen
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PCT/US1992/002888
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English (en)
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Sandra Sulikowski Fenton
Tammy Browne Strassburg
Sheryl Sanford Sullivan
Robert William Zercie
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Eastman Kodak Company
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Priority to JP92508868A priority Critical patent/JPH05507095A/ja
Publication of WO1992018535A1 publication Critical patent/WO1992018535A1/fr
Priority to KR1019920703159A priority patent/KR930700548A/ko
Priority to FI925593A priority patent/FI925593A/fi

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    • 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

  • This invention relates to the detection of endometriosis, and to protein antigenic fragments, reagents, diagnostic test kits and compositions useful therein.
  • Endometriosis is a disease state in which tissues resembling the uterine mucous membrane, or endometrium (located in the lining of the uterus) , multiply in other parts of the body, such as in the abdominal cavity. This disease is a significant problem in gynecology. The presence of the abnormal tissues can cause abdominal bleeding, adhesions, dysmenorrhea and particularly infertility.
  • 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 present invention provides a protein antigen isolated from the cytoplasm of epithelial adenocarcinoma cells, the antigen selected from the group consisting of: a. a fragment having a molecular weight of from about 63 to about 67 kilodaltons, b. a fragment having a molecular weight of from about 33 to about 37 kilodaltons, c. a fragment having a molecular weight of from about 40 to about 44 kilodaltons d. a fragment having a molecular weight of from about 31 to about 35 kilodaltons, and e. a fragment having a molecular weight of from about 57 to about 64 kilodaltons.
  • antigens can be provided individually or in admixture in a buffered composition useful for detecting the presence of endometrial antibodies.
  • This invention also provides an endometrial antibody capture reagent comprising one or more of the protein endometrial antigens described above attached to a water insoluble support. Additionally, one or more of the antigens can be detectably labeled to provide water soluble endometrial antibody detection reagents.
  • a method for determining endometriosis comprises:
  • the present invention provides an advantageous means for detecting endometriosis without the need for invasive laparoscopy. This result is achieved using novel protein antigen fragments isolated from the cytoplasm of epithelial adenocarcinoma cells to detect the presence of endometrial antibodies in a patient specimen, such as serum. Sensitive detection of the antibodies can be carried out using various assay formats, as described below. Brief Description of the Fi ⁇ ure The FIGURE is a photographic image of several nitrocellulose strips used in an immunoblot assay, as described in more detail in Example 5 below. Detailed Description of the Invention
  • the antigens of this invention are identified generally by molecular weight in kilodaltons. They are identified in a narrow range of molecular weights since it is standard in the art to have some inherent inaccuracy (about 10%) in electrophoretic methods for molecular weight determination. Some of the antigens have also been characterized by isoelectric point (pi) .
  • the antigens are generally protein fragments isolated from larger proteins found in the cytoplasm of human epithelial adenocarcinoma cells. Included among such cells are endometrial, breast and ovary cells.
  • the antigen fragments identified herein can be isolated from various cell lines, and may have varying amino acid compositions even though the molecular weight is the same. Also contemplated as equivalents of the naturally occurring antigens isolated from cells are what are termed "immunological equivalents" which are peptides which have the same molecular weight, isoelectric point and immunological reactivity with the antibodies of interest.
  • HEC1A 63-67 (ATCC HTB-112) 33-37 40-44 59-64
  • Endometrial antigens can be isolated by affinity chromatography of extracts of epithelial tissue (such as endometrial tissue) which has been subjected to extraction reagents such as detergents.
  • Antibodies monoclonal or polyclonal
  • the treatment of the tissue extracts by column purification can be carried out using standard procedures, for example, those described by Davis et al, Cane. Res. 46. pp. 6143-6148 (1986) .
  • isolated is meant that the protein antigen fragment is in an at least partially purified state compared to its natural state in the cytoplasm of the epithelial adenocarcinoma cells, or in any other human fluid or tissue specimen.
  • Various techniques besides those described herein can be used to isolate the individual antigen fragments including electroelution from gels as described, for example, by Harrington, Methods of Enzvmolocrv 1£2, pp. 488-495 (1990).
  • the antigens can be obtained from tissue culture cells such as cultures of epithelial adenocarcinoma cell lines such as those mentioned in EP-A-0 387 027. Representative useful cell lines are on deposit with the American Type
  • HEC1A ATCC HTB-112
  • AN3CA ATCC HTB-111
  • RL95- 2 ATCC CRL-1671
  • KLE ATCC CRL-1622
  • T47D ATCC HTB- 133
  • CAOV3 CAOV3
  • the general procedure for isolating the antigens from a cell line is as follows: The cells are grown in the recommended medium to greater than 90% confluency, followed by homogenization in buffered saline solution or a solution of tris(hydroxymethyl)- aminomethane, sucrose and protease inhibitor. For extracts in buffered saline solution, particulate material is removed by centrifugation and the supernatant concentrated. In a preferred embodiment, for extracts in the sucrose solution, particulate materials are removed by centrifugation and the supernatant is spun at 100,000 x gravity for 1 hour at 4°C, then concentrated.
  • Extracts that is, the cytoplasmic fraction
  • SDS-PAGE electrophoresis for an appropriate time and voltage in an appropriate buffered system.
  • the resulting proteins are then transferred to nitrocellulose using standard im unoblotting techniques described, for example, by
  • 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.
  • the antigens described herein can also be provided as detectably labeled water soluble (or water suspendible) reagents which have an appropriate label moiety coupled thereto.
  • Useful labels include those directly detectable, such as radioisotopes, chromogens, fluorogens, suspendible magnetic particles, suspendible dyed polymeric particles, chemiluminescent moieties, bioluminescent moieties, phosphors and others known in the art.
  • Labels which are indirectly detectable through reaction with additional reagents include enzymes, dye-formers and others known in the art (for example, in EP-A-0 387 027).
  • Particularly useful labels include radioisotopes and enzymes.
  • 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 label moieties can be coupled to antigen fragments using standard technology described, for example, in US-A-4,302,438, Marchalonis, Biochem. J.. 113. pp. 299-305 (1969) Hnatowich et al, J.Immunol. Methods. £ ⁇ . t pp. 147-157 (1983) and Science, 220. pp. 613-615 (1983) for radiolabeling, and Yoshitake et al, Eur.J.Biochem. , 101. 395 (1979), Pesce et al, Clin.Chem.. 2__>, pp.
  • Antigens can be coupled to magnetic or magnetizable particles using the teaching of, for example, US-A-4,795,698.
  • Chemiluminescent moieties can be coupled to antigens according to the teaching of, for example, US-A-4,380,580. Dyed or fluorescent particles are useful as labels and can be attached to antigen according to US-A-4,259,313, EP-A-0 208 556 and EP-A-308 235. Fluoroscein or other fluorescent moities can be attached as a label using known procedures.
  • the antigen can also be labeled with a specific binding moiety that is not specific for endometrial antibodies.
  • moieties include avidin, biotin, a lectin, a sugar and others readily apparent to one skilled in the art.
  • the moiety would be reactive with its corresponding receptor which can be labeled with an enzyme radioisotope or other moiety as described above.
  • the antigen is labeled with biotin
  • the corresponding receptor, avidin can be coupled to an enzyme.
  • Such labeling techniques are described, for example, in US-A- 4,496,654, EP-A-0 201 079 and EP-A-0 370 694.
  • particles used in this reagent to be water suspendible normally they are less than about 1 m eters in size so that they stay suspended in water for at least 3 hours with little or no agitation.
  • the antigen of this invention can also be coupled with water insoluble supports to provide endometrial antibody capture reagents for complexing with the endometrial antibody, thereby "capturing" the antibody- Any useful support can be used as long as it is not readily suspendible in water (unlike the reagents described above) and does not interfere with the antibody-antigen reaction or any other reactions necessary for detection of that immunological reaction.
  • Useful supports include particles of organic and inorganic polymers, glass, ceramics, silica gel, metals, metal oxides, filters of paper, glass, matted fibers and particulate structures, microporous polymeric filters, gels, microtiter plates, test tubes, test cups, vials and others readily apparent to one skilled in the art.
  • 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.
  • Antibodies to the antigens of this invention can be developed using standard technology.
  • polyclonal antibodies can be prepared using suitable mammals, such as goats, monkeys, rabbits, guinea pigs and horses.
  • the resulting antisera can be purified using conventional affinity chromatography such as described by Mishell et al. Selected Methods in Cellular Immunology. San Francisco, Freeman (1980) .
  • 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.
  • Detection of endometrial antibodies can be carried out using a variety of immunological methods, all of which are generally well known in the art as involving the preferential binding of the antigens of this invention with the corresponding endometrial antibodies.
  • Such methods include, but are not limited to, competitive binding assays, enzyme-linked im unosorbent immunoassays (ELISA) , radioimmunoassays (R ⁇ A) , immunometric assays (sandwich), immunoblots, agglutination assays, light scattering assays and ultrasonic probe assays.
  • Immunoblots can be carried out using standard procedures described, for example, by Stott (noted above) .
  • the antigen is transferred to an immunoblot medium such as nitrocellulose (which is preferred), nylon or polyvinylidine difluoride, nonspecific sites are blocked with appropriate materials, and the patient sample is brought into contact with the medium for a sufficient period of time and temperature for antibodies in the sample to complex with antigen in the medium.
  • the complex in the medium can be contacted with detectably labeled antigen which can "sandwich" the antibodies, or with detectably labeled anti-human antibodies which are reactive with the endometrial antibodies.
  • a representative immunoblot is described in Example 5 below, and the results shown in the Figure.
  • Light scattering assays are useful to detect endometrial antib*_ dies using the antigens of this invention according to the teaching of EP-A-0 387 027.
  • Competitive binding assays generally involve contacting the specimen suspected of containing endometrial antibodies with the antigen and a known quantity of labeled endometrial antibodies.
  • the labeled and unlabeled antibodies compete to complex with the antigen, and the amount of detectable signal from the complex is inversely proportional to the amount of unlabeled antibodies in the specimen.
  • the labeled antibodies can be prepared using materials and procedures described generally above for labeled antigen. Further details of such assays can be obtained by consulting the considerable literature in this art, including US-A-3,654,090.
  • Another type of immunoassay is what is known as an immunometric or sandwich assay in which the targeted endometrial antibodies are "sandwiched" between specific binding materials.
  • the specific binding materials both comprise endometrial antigen, one being detectably labeled (that is a detection reagent) , and the other being a capture reagent as described above.
  • one specific binding material in the sandwich can be either a capture or detection reagent comprising an antigen as described herein, and the other is a capture or labeled anti-human antibody reactive with the endometrial antibodies.
  • the anti-human antibodies are advantageously labeled with a fluorogen, enzyme or radioisotope.
  • the assay is Icnown as an ELISA. Specific details about such assays are well known in the art, including US-RE-32,696, US-A-4,376,110 and US- A-4,486,530.
  • Anti-human antibodies reactive with the endometrial antibodies can be prepared using standard techniques. Many are commercially available.
  • Endometrial antibodies can be isolated from serum or other patient specimens by transferring an antigen fragment of this invention to nitrocellulose, contacting the immobilized antigen with the patient sample to complex the endometrial antibodies, and eluting the antibodies off the nitrocellulose using a high salt or low pH solution according to standard procedures.
  • test devices include micro-titer plates and disposable test devices (commercially available in SURECELLTM test kits marketed by Eastman Kodak Company) having microporous membrane disposed therein for separating complexed materials from uncomplexed materials.
  • 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.
  • Example 1 Isolation of Endometrial Antigens The following procedure and materials were used to isolate several antigen fragments using various epithelial adenocarcinoma cell lines.
  • HEC1A ATCC HTB-112
  • AN3CA ATCC HTB-111
  • RL95-2 ATCC CRL-1671
  • KLE ATCC CRL- 1622
  • T47D ATCC HTB 133
  • CAOV3 ATCC HTB-75
  • Each cell line was treated in the following manner: it was grown in recommended media (for example, commercially available McCoy's media for HEC1A) to greater than 90% confluency (at this level, there is confidence that one or more of the antigens are present) .
  • recommended media for example, commercially available McCoy's media for HEC1A
  • the resulting cells were homogenized in a solution of tris(hydroxymethyl)aminomethane buffer (50 mmolar, pH 7.4), sucrose (250 mmolar) and protease inhibitor (a mixture of 0.5 ⁇ g/ml of leupeptin, 0.7 ⁇ g/ ⁇ l of pepstatin, 372 ⁇ g/ml of EDTA Na2 and 2 ⁇ g/ml of aprotinin available from Boehringer-Mannheim or Sigma Chemical) for 2 minutes at 4°C using a mechanical homogenizer.
  • protease inhibitor a mixture of 0.5 ⁇ g/ml of leupeptin, 0.7 ⁇ g/ ⁇ l of pepstatin, 372 ⁇ g/ml of EDTA Na2 and 2 ⁇ g/ml of aprotinin available from Boehringer-Mannheim or Sigma Chemical
  • Cellular debris including nuclei, mitochondria and unlysed cells, was removed by centrifugation, followed by ultracentrifugation at 100,000 x gravity for 1 hour at 4°C to remove all unwanted cellular debris and leaving the contents of the cytoplasm. The supernatant was then concentrated using a Centricell concentrator (30,000 normal molecular weight limit, Polysciences, Warrington, Pa.) to about 0.5-10 g/ml protein.
  • Centricell concentrator 30,000 normal molecular weight limit, Polysciences, Warrington, Pa.
  • 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 proteins were blotted to nitrocellulose as described in Stott, J.Immun. Methods. 119, pp. 153-187, (1989). The blotted protein was probed with patient serum. Sera believed to contain endometrial antibodies (as determined by indirect immunofluorescence microscopy) and as being from patients known to have endometriosis (as determined by laparoscopy) were tested. Sera known to be negative for endometrial antibodies using similar techniques were also tested as negative controls. Reactive proteins were developed using a standard detection system.
  • 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 molecular weight of each fragment was determined by comparing measured distances (cm) of target antigens to the measured distances (cm) of a resolved mixture of standard SDS-PAGE low molecular weight protein markers. Molecular weights were determined and expressed in kilodaltons (kD) using a polynomial curve fit calculated using CRICKETGRAPH TM software (available from Cricket Software, Inc.), and EXCEL TM software (available from Microsoft) . Both programs were run on a Macintosh 2 computer.
  • 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 antigens were was further purified by anion-exchange chromatography using a Waters PROTEIN- PAi ⁇ TM DEAE column.
  • the antigen fragments were then eluted from the column using tris(hydroxymethyl)amino ⁇ methane buffer (0.02 molar, pH 8) and a sodium chloride gradient.
  • the antigens eluted at between 0.3 and 0.45 molar sodium chloride.
  • 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.
  • Serum samples were diluted at 1:5 or 1:10 in a diluent of TWEENTM 20 (0.05%) and bovine serum albumin (3%) in phosphate buffered saline solution. The diluted samples were incubated in the plate wells for two hours at room temperature while being shaken. The wells were washed three time with the wash solution containing TWEENTM 20.
  • the plate was again washed three times, and a dye-providing composition (200 ⁇ l) was added to each well and allowed to react for about five minutes.
  • This composition included 2-(4-hydroxy-3-methoxyphenyl)-4,5- bis(4-methoxyphenyl)imidazole leuco dye (0.2 mmolar) pol (vinylpyrrolidone) (1.25 %) , 4'-hydroxyacetanilide (5 mmolar) , diethylenetriaminepentaacetic acid chelator (0.01 mmolar) and hydrogen peroxide (8 mmolar) in sodium phosphate buffer (10 mmolar, pH 6.8).
  • Example 2 demonstrates the use of the isolated endometrial antigens to detect endometrial antibodies present in patient blood sera by immunoblot ing techniques.
  • the antigen fragments described in Example 1 were transferred to nitrocellulose strips using optimized immunoblotting techniques as described by Stott, supra.
  • the buffer for transfer was composed of tris(hydroxymethyl)aminomethane buffer (25 mmolar, pH 8.3), glycine (200 mmolar) and methanol (20%, HPLC grade) . To carry out the transfer, 75 volts at 4°C were applied for 2 hours.
  • 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 strips were then incubated for two hours at 24°C in contact with anti-human antibodies (13.2 ⁇ l of conjugate in 40 ml of solution used to dilute patient serum) directed to the serum endometrial antibodies bound to the immobilized transferred antigen fragments.
  • the anti-human antibodies were comprised of goat anti-human IgG (heavy and light chain) antibodies labeled with alkaline phosphatase for detection.
  • the conjugate was purchased as part of an IMMUN-BLOT TM assay kit (BioRad Laboratories) .
  • 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.
  • the FIGURE shows the immunoblot bands in strips la, lb, 2a and 2b for the C3 (40-44 kilodalton) and C2 (33-37 kilodalton) fragments (identified in the
  • 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.
  • Example 6 Detection of Endometrial Antibodies Using Disposable Test pevice This example demonstrates the detection of endometrial antibodies using a disposable test device and the ELISA immunological technique. Materials:
  • Non-purified antigen (containing fragments Ci, C2 and C3, identified in Table I above) isolated from HEC1A cell line (as described above) was covalently attached to particles composed of poly[styrene-jS ⁇ j-3-(p_-vinylbenzyl-thio)propionic acid] (97.59:2.41 molar ratio, 1.4 mm average diameter) (2.53% solids) to form a particulate endometrial antibody capture reagent of this invention.
  • a particulate negative control reagent was prepared by similarly immobilizing ⁇ -casein on the same type of particles (2.05% solids).
  • the disposable test device used was a SURECELL TM test device (Eastman Kodak Company) having a LOPRODYNE TM polyamide icroporous (5 ⁇ ) membrane (Pall
  • the test device has three test wells, one for the negative control, and two for the specimen.
  • the particulate reagents described above (0.3% final solids) were coated on designated test well membranes in the test device, and dried overnight at room temperature.
  • the serum diluent composition used was composed of tris(hydroxymethyl)aminomethane (100 mmolar, pH 8.0), succmylated casein (1%), TWEEN TM 20
  • the wash solution was comprised of 1-methyl-
  • TM chloride 500 mmolar
  • TWEEN 20 0.25%
  • sodium phosphate sodium phosphate
  • monobasic 50 mmolar, pH 7.4
  • the anti-human antibodies used were goat anti-human IgG (heavy and light chain) antibodies and were conjugated with horseradish peroxidase (available from Jackson Immunoresearch) .
  • the leuco dye composition comprised 2-(4- hydroxy-3-methoxyphenyl)-4,5-bis(4- methoxyphenyl)imidazole leuco dye (0.23 mmolar) poly(vinylpyrrolidone) (1.25 %) , 4'-hydroxyacetanilide (5 mmolar) , diethylene-triaminepentaacetic acid chelator (0.01 mmolar) and hydrogen peroxide (8 mmolar) in sodium phosphate buffer (10 mmolar, pH 6.8).
  • the dye stop solution comprised sodium azide (0.1%) .
  • a patient serum sample (25 ⁇ l) was diluted by adding it to the serum diluent (2 ml) in standard assay squeeze tubes. Filter tips were attached to the tubes, and the diluted sample was added to each well of the test device to the top of the fill dot. Fluid was allowed to flow through the membranes.
  • 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.
  • the visual dye signal was evaluated and scored (0 being lowest dye signal and 10 being the highest) . Transmission densities were also measured using standard densitometric procedures. The results are shown in Table V below.
  • Serum Sample known to be positive for antibodies

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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.
PCT/US1992/002888 1991-04-09 1992-04-09 Antigene endometrial, composition, trousse pour epreuve et methode de determination d'anticorps endometriaux WO1992018535A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP92508868A JPH05507095A (ja) 1991-04-09 1992-04-09 子宮内膜抗原、組成物、試験キット及び子宮内膜抗体検出方法
KR1019920703159A KR930700548A (ko) 1991-04-09 1992-12-09 자궁내막 항체 측정방법 및 측정용 자궁내막 항원, 조성물, 테스트킷
FI925593A FI925593A (fi) 1991-04-09 1992-12-09 Endometrial antigen, komposition, testfoerpackning och foerfarande foer bestaemning av endometrial antikropp

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US68217791A 1991-04-09 1991-04-09
US682,177 1991-04-09

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JP (1) JPH05507095A (fr)
KR (1) KR930700548A (fr)
CA (2) CA2062675A1 (fr)
FI (1) FI925593A (fr)
IE (1) IE921132A1 (fr)
WO (1) WO1992018535A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994028021A1 (fr) * 1993-05-28 1994-12-08 Medical University Of South Carolina Proteines endometriales, compositions antigeniques et procedes de detection de l'endometriose
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
WO1999000671A2 (fr) * 1997-06-26 1999-01-07 The Regents Of The University Of Michigan Procede d'identification d'antigenes de proteines cellulaires et de detection de la presence d'anticorps diriges contre des proteines cellulaires specifiques du serum
WO2000026668A2 (fr) * 1998-11-05 2000-05-11 The Regents Of The University Of Michigan Proteines s100 et auto-anticorps comme marqueurs seriques pour le cancer
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
US6743595B1 (en) 1999-01-25 2004-06-01 Metriogene Biosciences Inc. Method and diagnostic kit for diagnosis of endometriosis
US7879562B2 (en) 2004-07-07 2011-02-01 Siemens Healthcare Diagnostics Inc. Methods of diagnosing endometriosis in human subjects using the ME-5 polypeptide

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0387027A2 (fr) * 1989-03-07 1990-09-12 Adeza Biomedical Corporation Méthodes et réactifs pour le diagnostic de l'endométriose

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0387027A2 (fr) * 1989-03-07 1990-09-12 Adeza Biomedical Corporation Méthodes et réactifs pour le diagnostic de l'endométriose

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AM. J. REPROD. IMMUNOL. MICROBIOL. vol. 13, 1987, pages 62 - 65; R. A. WILD ET AL.: 'Epithelial localization of antiendometrial antibodies' *
FERTILITY AND STERILITY vol. 50, no. 6, 1988, pages 860 - 863; S. MATHUR ET AL.: 'Endometrial antigens' *
FERTILITY AND STERILITY vol. 55, no. 1, 1991, pages 90 - 94; R. A. WILD ET AL: 'Endometrial antibodies versus CA125' *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994028021A1 (fr) * 1993-05-28 1994-12-08 Medical University Of South Carolina Proteines endometriales, compositions antigeniques et procedes de detection de l'endometriose
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
WO1999000671A2 (fr) * 1997-06-26 1999-01-07 The Regents Of The University Of Michigan Procede d'identification d'antigenes de proteines cellulaires et de detection de la presence d'anticorps diriges contre des proteines cellulaires specifiques du serum
WO1999000671A3 (fr) * 1997-06-26 1999-06-10 Univ Michigan Procede d'identification d'antigenes de proteines cellulaires et de detection de la presence d'anticorps diriges contre des proteines cellulaires specifiques du serum
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
WO2000026668A2 (fr) * 1998-11-05 2000-05-11 The Regents Of The University Of Michigan Proteines s100 et auto-anticorps comme marqueurs seriques pour le cancer
WO2000026668A3 (fr) * 1998-11-05 2000-09-08 Univ Michigan Proteines s100 et auto-anticorps comme marqueurs seriques pour le cancer
US6743595B1 (en) 1999-01-25 2004-06-01 Metriogene Biosciences Inc. Method and diagnostic kit for diagnosis of endometriosis
US7879562B2 (en) 2004-07-07 2011-02-01 Siemens Healthcare Diagnostics Inc. Methods of diagnosing endometriosis in human subjects using the ME-5 polypeptide
US7981626B2 (en) 2004-07-07 2011-07-19 Siemens Healthcare Diagnostics Inc. Method of detecting endometriosis in human subjects using SEQ ID No. 9 or an epitope thereof

Also Published As

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FI925593A0 (fi) 1992-12-09
FI925593A (fi) 1992-12-09
JPH05507095A (ja) 1993-10-14
CA2062675A1 (fr) 1992-10-10
IE921132A1 (en) 1992-10-21
KR930700548A (ko) 1993-03-15
EP0538430A1 (fr) 1993-04-28
CA2081900A1 (fr) 1992-10-10

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