IE921132A1 - Endometrial antigen, composition, test kit and method for¹endometrial antibody determination - Google Patents

Abstract

A plurality of protein antigen fragments have been isolated from the cytoplasm of epithelial adenocarcinoma cells. The protein antigens are useful in the detection of endometrial antibodies which are indicative of endometriosis. The antigens can be attached to water insoluble supports or detectably labeled to form reagents. Detection of endometrial antibodies is accomplished by reacting the antigen with the antibodies in a specimen sample followed by detection of the resulting complex. The antigens can be supplied as a buffered composition in a diagnostic test kit.

Description

ENDOMETRIAL ANTIGEN, COMPOSITION, TEST KIT AND METHOD FOR ENDOMETRIAL ANTIBODY DETERMINATION 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.

Currently, a preliminary diagnosis of endometriosis is generally made based on a patient's history of infertility, unexplained pelvic pain or other known symptoms. Confirmation is carried out using a surgical procedure termed laparoscopy to obtain a sample of tissue for biopsy. This is a procedure which is unpleasant as well as having the usual dangers associated with invasive procedures.

It has been reported that antibodies to normal endometrial tissues have been found in the serum of patients with endometriosis. Various antigens have been speculated as immunologically related to the endometrial antibodies found in the serum specimens.

In EP-A-0 387 027, 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. -2It would be desirable to have additional antigen fragments 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 characterized as being: a. a fragment having a molecular weight of from 63 to 67 kilodaltons, b. a fragment having a molecular weight of from 33 to 37 kilodaltons, c. a fragment having a molecular weight of from 40 to 44 kilodaltons d. a fragment having a molecular weight of from 31 to 35 kilodaltons, or e. a fragment having a molecular weight of from 57 to 64 kilodaltons. The invention also provides a buffered antigenic composition useful for detecting the presence of endometrial antibodies comprising a protein antigen isolated from the cytoplasm of epithelial adenocarcinoma cells, the composition characterized wherein the protein antigen is one of those a. through e. described above.

This invention also provides an endometrial antibody capture reagent comprising a water insoluble support, the reagent characterized as having one or more of the protein antigens a. through e. described above attached to the water insoluble support.

The invention also provides a water soluble endometrial antibody reagent comprising a protein antigen which is detectably labeled, the reagent characterized wherein the antigen is one of those a. through e. described above.

The invention also provides a diagnostic test kit comprising: -31) an antigen a. through e. described above, and 2) an anti-human antibody reactive with an endometrial antibody.

The invention also provides a method for detecting endometrial antibodies comprising: A. contacting a specimen suspected of containing endometrial antibodies with a protein antigen as described above, and B. detecting any resulting complex of the antigen with the specimen endometrial antibodies.

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.

The FIGURE is a photographic image of several nitrocellulose strips used in an immunoblot assay, as described in more detail in Example 4 below.

The antigens of this invention are identified generally by molecular weight in kilodaltons. They are identified in a narrow range of molecular weight since it is standard in the art to have some inherent inaccuracy ( 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 -4acid 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.

Representative isolated fragments of this invention are listed in Table I below, and can be used singly or in mixtures in the practice of this invention .

TABLE 1 Cell Line_Antigen Fragment_Molecular Weight-(kDL RL95-2 A 63-67 (ATCC CRL-1671) AN3CA Βχ (ATCC HTB-111) B2 ' B3 B4 HEC1A Ci (ATCC HTB-112) C2 C3 C4 63-67 33-37 40-44 59-64 63-67 33-37 40-44 59-64 KLE D (ATCC CRL-1622) T47D El (ATCC HTB-133) e2E3 e4 e5 31-35 63-67 33-37 40-44 59-63 57-59 CAOV3 Fl 63-67 (ATCC HTB-75) ^2 33-37 40-44 59-64 It is preferred to use the 33-37 kD, 40-44 kD and 57-59 kD fragments noted above, individually or in a mixture .

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) specific to the antigens can be used in the chromatography process.

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). By isolated is meant that the 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 human fluid or tissue specimen.

In a preferred process, the antigens can be obtained from tissue culture cells such as cultures of epithelial adenocarcinoma cell lines for example those mentioned in EP-A-0 387 027. Representative useful cell lines are on deposit with the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland 20852, USA), namely: cell lines HEC1A (ATCC HTB-112), AN3CA (ATCC HTB-111), RL95-2 (ATCC CRL-1671), KLE (ATCC CRL-1622), T47D (ATCC HTB-133) and CAOV3 (ATCC HTB-75).

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% -6confluency, 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 (cytoplasmic fraction) are then resolved using SDS-PAGE electrophoresis for an appropriate time and voltage in an appropriate buffer system. The resulting proteins are then transferred to nitrocellulose using standard immunoblotting techniques.

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 6 to 8 using one or more suitable buffers such as phosphate buffered saline solution, tris(hydroxymethyl)aminomethane, glycine, 3(N-morpholino)propanesulfonic acid and borates. 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 -7through reaction with additional reagents include enzymes, dye-formers and others known in the art (for example, in EP-A-0 387 027). Useful enzyme labels include peroxidase, alkaline phosphatase, urease, glucose oxidase and beta-aalactosidase.

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. £5, pp. 147-157 (1983) and Science, 220f pp. 613-615 (1983) for radiolabeling, and Yoshitake et al, Eur.J.Biochem., 101, 395 (1979), Pesce et al, Clin.Chem.. 2£, pp. 353-359 (1974), US-A4,302,438, US-A-4,376, 110 and US-RE-31,006 for labeling with enzymes. Antigens can be coupled to magnetic or magnetizable particles according to US-A-4,795,698. Chemiluminescent moieties can be coupled to antigens according to the teaching of, for example, US-A4,380,580. Dyed or fluorescent particles are useful as labels and can be attached to antigen according to USA-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. Such 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.

For particles used in this reagent to be water suspendible, normally they are less than 1 pmeter in size so that they stay suspended in water for at least 3 hours with little or no agitation. -8The 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 and vials. The particles are generally greater than 0.05 pmeters in diameter. The antigens can be attached to such materials by adsorption or other non-covalent means or by covalent means using techniques generally known.

Antibodies to the antigens of this invention can be developed using standard technology. For example, polyclonal antibodies can be prepared using suitable mammals as hosts. The resulting antisera can be purified using conventional affinity chromatography.

Non-human monoclonal antibodies can also be prepared using the standard method of Kohler et al, Nature, 256. pp. 495-497 (1975) involving the use of suitable hybridomas prepared from immunized mice or rats to produce suspended spleen cells.

The antigens of this invention are useful for the detection 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. Preferably, the antibodies are detected in blood serum. These antibodies are generally identified as human IgG type -9antibodies 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 immunosorbent immunoassays (ELISA), radioimmunoassays (RIA), immunometric assays (sandwich), immunoblots, agglutination assays, light scattering assays and ultrasonic probe assays.

Immunoblots can be carried out using standard procedures. Generally, 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. Following washing, the complex in the medium can be contacted with detectably labeled antigen which can sandwich the antibodies, or with detectably labeled anti-antibodies directed to the endometrial antibodies.

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. In one embodiment, 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. In another embodiment one specific binding material in the sandwich can be either a capture or detection reagent comprising an antigen as -10described 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.

Patient samples, such as serum samples, can be diluted if desired with water, buffer or suitable diluents commercially available for that purpose. Particularly useful diluent compositions are described in EP-A-0 337 785.

The assays can be carried out in appropriate equipment or test devices. Immunoblots, for example, are carried out using appropriate media, such as nitrocellulose strips. Competitive binding and sandwich assays can be carried out using micro-titer plates having a multiplicity of test wells, test tubes, test slides and disposable test devices. Preferred test devices include microtiter plates and disposable TM test devices (commercially available m SureCell test kits marketed by Eastman Kodak Company) having microporous membrane disposed therein for separating complexed materials from uncomplexed materials.

The antigens, compositions or reagents of this invention can be supplied individually or as part of a diagnostic test kit. Such 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. Preferably, the kit includes the antigen (or mixture thereof), labeled anti-antibodies to 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. -11The following examples are presented here to illustrate the practice of this invention. Unless otherwise indicated, the percentages are by weight. Example 1: Isolation of Endometrial Antigens The following procedure and materials were used to isolate several antigen fragments using various epithelial adenocarcinoma cell lines.

Six cell lines: HEC1A (ATCC HTB-112), AN3CA (ATCC HTB-111), RL95-2 (ATCC CRL-1671), KLE (ATCC CRL10 1622), T47D (ATCC HTB 133) and CAOV3 (ATCC HTB-75) were obtained from the American Type Culture Collection.

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). The resulting cells were homogenized in a solution of tris(hydroxymethyl)amino-methane buffer (50 mmolar, pH 7.4), sucrose (250 mmolar) and protease inhibitor (a mixture of 0.5 pg/ml of leupeptin, 0.7 pg/ml of pepstatin, 372 pg/ml of EDTA Na2 and 2 pg/ml of aprotinin available from Boehringer-Mannheim or Sigma Chemical) for 2 minutes at 4°C using a mechanical homogenizer. 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 0.5-10 mg/ml protein.

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 -12buffer (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 two5 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 SDS10 PAGE electrophoresis in the second dimension. The proteins were blotted to nitrocellulose. 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 -13polynomial curve fit calculated using Cricketgraph software (available from Cricket Software, Inc.), and TM EXCEL software (available from Microsoft). Both programs were run on a Macintosh 2 computer. 10 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 known procedures and a 10% accepted in the art. difficult to determine using variation is generally TAB L Ε II Antigen Fragment Cell Line Source Molecular Isoelectric Weight (kD)** Point @ A RL95-2 63-67 NA* 15 Bl AN3CA 63-67 NAb2 AN3CA 33-37 NAb3 AN3CA 40-44 NA b4 AN3CA 59-64 NA Cl HEC1A 63-67 4.5 20 C2 HEC1A 33-37 5.8c3 HEC1A 40-44 4.6 C4 HEC1A 59-64 6.0 Ei T47D 63-67 4.5 E2 T47D 33-37 NA 25 E3 T47D 40-44 4.6 e4 T47D 59-64 NA E5 T47D 57-59 NA El CAOV3 63-67 4.5 F2 CAOV3 33-37 5.5 30f3 CAOV3 40-44 4.6 f4 CAOV3 59-64 6.0 35 *NA = not available ** kD = kilodaltons @ values reflect plus or minus 1.0 -14Example 2: Buffered Compositions-of Antigens One or more of the antigens were added to tris(hydroxymethyl)aminomethane buffer (pH 8.3) to form a crude buffered composition of this invention. These compositions can be stored in suitable containers, for example in test kits, until their use or immobilization on solid supports for use in assays.

Example 3: Preparation of Endometrial Antibody.

Capture Reagent This is an example of the preparation of an endometrial antibody capture reagent of this invention.

A mixture of endometrial antigens (Εχ, E2/ e 4 and E5) were isolated as described in Example 1.

The antigen fragments were further purified 15 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)amino methane buffer (pH 7.5). This suspension was dialyzed against the buffer.

The antigens were further purified by anionexchange chromatography using a Waters PROTEIN-PAK™ DEAE column. The antigen fragments were then eluted from the column using the buffer (0.02 molar, pH8) and a sodium chloride gradient. The antigens eluted from the column at between 0.3 and 0.45 molar sodium chloride .

The resulting solution was diluted to 30 gg protein/ml in phosphate buffered saline solution, containing protease inhibitors and a sample (100 gl) was added to each well of a polystyrene microtiter plate and incubated at room temperature for two hours. -15The 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 Tween™ 20 nonionic surfactant (0.05%) in phosphate buffered saline solution.

Serum samples were diluted at 1:5 or 1:10 in a diluent of Tween™ 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 Tween™ 20.

Goat anti-human immunoglobumin F(ab')2 fragments conjugated to horseradish peroxidase, diluted in the diluent and filtered through a 0.2 micrometer filter, were added to the wells and allowed to react for 1 hour at room temperature.

The plate was again washed three times, and a dye-providing composition (200 μΐ) was added to each well and allowed to react for about five minutes. This composition included 2-(4-hydroxy-3-methoxyphenyl)-4, 5bis(4-methoxyphenyl)imidazole leuco dye (0.2 mmolar) poly(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).

A solution (100 μΐ) to stop dye formation was added and the dye density was evaluated. The test samples were evaluated using a calibration curve generated from known positive and negative serum samples . -16Example 4: Detection of Endometrial Antibodies in Patient- Specimens This example demonstrates the use of the isolated endometrial antigens to detect endometrial antibodies present in patient blood sera by immunoblotting 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. Upon completion of the transfer, 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 TM 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), and Tween 20 nonionic surfactant (0.05%).

Sera believed to contain endometrial antibodies (as determined by indirect immunofluor 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. Sera was also tested without prior -17knowledge of the presence or absence of endometriosis. These tests are identified in Table IV below with *.

After serum incubation, the nitrocellulose strips were washed four times (5 minutes each time) with the buffered solution containing Tween 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 μΐ of conjugate in 40 ml of solution used to dilute patient serum) which were reative with 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 tm ... as part of an IMMUN-BLOT assay kit (BioRad Laboratories). Upon completion of the antibodyconjugate incubation, the nitrocellulose strips were washed four times (5 minutes each) with the buffered TM Tween 20 solution noted above, and once (5 minutes) TM with the buffer solution noted above without Tween 20 to remove uncomplexed reactants and excess Tween 20 nonionic surfactant.

To detect the resulting bands in the strips, a dilute solution of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium [500 μΐ of each reagent stock in 50 ml of 0.1 molar tris(hydroxymethyl) aminomethane buffer, pH 9.5] were added.

Upon completion of sufficient color development of the bands (usually 20 minutes), the substrate was removed and the strips were washed with deionized, distilled water for 10 minutes to quench further color formation. 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 -18Table 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. Antigen Fragment(s) TABLE III Normal Patients (positives/ total samples) Endometriosis Patients (positives/ total samples) A 0/2 3/3 Bl 0/2 3/3 B2 0/2 3/3 Cl 1/8 9/11 c2 0/8 9/11 d2 0/2 2/3 TABLE IV Normal Patients Endometriosis Patients Antigen (positives/ (positives/ Fragment(s) total samples) total samples) * c2 2/8 20/26 * c3 2/8 19/26 * c4 1/10 14/16 * e2 0/5 5/5 * e3 0/5 5/5 f2 0/5 5/5 e3 0/5 5/5 B2 0/4 6/6 B3 0/4 6/6 b4 0/7 13/16 -19Example 5: Detection of Endometrial Antibodies Using Disposable last Device This example demonstrates the detection of 5 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 ί styrene-co-3-(β-vinylbenzylthio)propionic acid] (97.59:2.41 molar ratio, 1.4 pm average diameter) (2.53% solids) to form a particulate capture reagent of this invention.

A particulate negative control reagent was prepared by similarly immobilizing α-casein on the same type of particles (2.05% solids).

The disposable test device used was a TM SureCell test device (Eastman Kodak Company) having a TM LoProdyne polyamide microporous (5 pm) membrane (Pall Corporation) mounted therein. 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 TM mmolar, pH 8.0), succinylated casein (1%), Tween 20 (0.05%) and gum arabic (1%).

The wash solution comprised l-methyl-2pyrrolidinone (10%), Nonidet™ P-40 (0.1%), sodium chloride (500 mmolar), Tween™ 20 (0.25%), sodium phosphate, monobasic (50 mmolar, pH 7.4). -20The anti-human antibodies were goat antihuman IgG (heavy and light chain) antibodies and were conjugated with horseradish peroxidase.

The leuco dye composition comprised 2-(45 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%) .

Assay Procedure: A patient serum sample (25 μΐ) was diluted by 15 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.

Diluted (1:10,000) labeled anti-human antibodies (40 μΐ) were added to each test well. Fluid was allowed to drain through the membranes and the test was incubated for one minute. Each test well was then washed twice (250 μΐ) with the wash solution.

The dye-providing composition (40 μΐ) 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 μΐ) was then added, followed by fluid drainage.

The visual dye signal was evaluated and scored (0 being the 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. -21W ni * * Φ Γ—I oj ε φ cn +J « , Φ H Φ 1-1 CL ε o in Φ Φ > S Ή 4-1 r—1 (β o Cn O Φ 4-1 2 c 4-1 ω Φ H Φ 1—1 r-H ε φ cn m 1—I o o Γ m o Φ r—i hj e; <ΰ cn φ φ > 2= •Η 4-J *—I (0 o cn l Φ 4-1 2 C o o O C o -H CO >i CO 4-1 ω Φ -H n o b •H 4-> C rO L O M-l Φ > Ή 4-> -H ω o (X Φ b O 4-1 q o c X Φ »—I Oj ε (0 cn ε L Φ cn co Φ Ή b ο b •Η 4-> G (0 Ο Ο Μ-4 Φ > Ή 4-> (0 Cn φ G Φ b Ο 4-1 C Ο C Si φ 1—I α ε φ w ε Ο Φ cn Identifies the number of test wells used in the assay, so that n=2 means that the results in the Table represent the average of two test well •Η Ή « * c ε ω r-l 4-> * * s co C IT3 r—1 C Φ 3 3 (0 Q CO (0 L -Η Φ Eh > OS

Claims (20)

Claims:

1. A protein endometriosis antigen isolated from the cytoplasm of epithelial adenocarcinoma cells, the antigen characterized as being: a. a fragment having a molecular weight of from 63 to 67 kilodaltons, b. a fragment having a molecular weight of from 33 to 37 kilodaltons, c. a fragment having a molecular weight of from 40 to 44 kilodaltons, d. a fragment having a molecular weight of from 31 to 35 kilodaltons, or e. a fragment having a molecular weight of from 57 to 64 kilodaltons.

2. The antigen as claimed in claim 1 wherein the 63-67 kD fragment is isolated from the RL95-2, AN3CA, HEC1A, T47D or CAOV3 cell line, the 33-37 kD fragment is isolated from the AN3CA, HEC1A, T47D or CAOV3 cell line, the 40-44 fragment is isolated from the AN3CA, HEC1A, T47D or CAOV3 cell line, the 31-35 fragment is isolated from the KLE cell line, and the 57-64 fragment is isolated from the AN3CA, HEC1A, T47D or CAOV3 cell line.

3. A buffered antigenic composition useful for detecting the presence of endometrial antibodies, the composition comprising a protein antigen isolated from the cytoplasm of epithelial adenocarcinoma cells, the composition characterized wherein the antigen is one of those claimed in claim 1.

4. An endometrial antibody capture reagent comprising a water insoluble support, the reagent characterized as having a protein antigen as claimed in claim 1 attached to the water insoluble support. -2310

5. The reagent as claimed in claim 4 wherein the support is a microtiter plate.

6. The reagent as claimed in claim 4 wherein the support is a particle.

7. A water soluble endometrial antibody reagent comprising a protein antigen which is detectably labeled, the reagent characterized as comprising a protein antigen as claimed in claim 1.

8. The reagent as claimed in claim 7 which is labeled with a fluorogen, radioisotope or enzyme.

9. A diagnostic test kit comprising: 1) a protein antigen as claimed in claim 1, and 2. ) an anti-human antibody reactive with an endometrial antibody.

10. The kit as claimed in claim 9 wherein the antigen is attached to a water insoluble support.

11. A method for detecting endometrial antibodies comprising: A. contacting a specimen suspected of containing endometrial antibodies with a protein antigen as claimed in claim 1, and detecting a complex of the antigen with the endometrial antibodies. The method as claimed in claim 11 wherein the antigen is attached to a water insoluble support, and the resulting complex is thereby insolubilized for detection. B.

12 .

13. The method as claimed in claim 11 wherein the complex is detected by reaction of said endometrial antibody with a detectably labeled anti-human antibody reactive with the endometrial antibody.

14. The method as claimed in claim 13 wherein the anti-human antibody is labeled with a fluorogen, radioisotope or enzyme.

-2415. The method as claimed in any one of claims 11 to 14 wherein the specimen is blood serum.

16. A protein endometriosis antigen substantially as described herein with reference to the examples and accompanying figure .

17. A buffered antigen composition comprising an antigen as defined in claim 1 or claim 16.

18. An endometrial antibody capture reagent having a protein antigen as defined in claim 1 or claim 16.

19. A diagnostic test kit comprising an antigen as defined in claim 1 of claim 16.

20. A method for detecting endometrial antibodies using an antigen as claimed in claim 1 or claim 16 substantially as described herein.