METHOD OF ASSAYING ADENOCARCINOMA ANTIGENS

Abstract: Adenocarcinoma antigens in serum samples are assayed immobilizing, on a solid phase, a first antibody which is a mixture of anti-human lung adenocarcinoma monoclonal anti­ body ALC-l86 and anti-human stomach adenocarcinoma mono­ clonal antibody AMC-382, allowing a human serum sample to react with the first antibody to bind adenocarcinoma antigens, allowing a second antibody which is a mixture of anti-human lung adenocarcinoma monoclonal antibody ALC-l86 and anti-human stomach adenocarcinoma monoclonal antibody AMC-382 each in an enzyme-labeled form to react with the adenocarcinoma antigens and thereafter determining the quantity of the thus-bound second antibody by measuring the activity of the labeling enzyme.

Description: BACKGROUND OF THE INVENTION Field of the Invention This invention relates to an improvement in the field of clinical diagnosis and provides a method of assaying adenocarcinoma antigens which is applicable to clinical diagnosis of human adenocarcinomas. Description of the Prior Art The anti-human lung adenocarcinoma monoclonal antibody ALC-186 (hereinafter referred to as "ALC-186" for short) is a monoclonal antibody prepared by the present inventors and a serodiagnostic system using the same can detect lung adenocarcinoma at a positive rate of 48% (European Patent Application No. 86 114 025.9 filed on October 9, 1986). The anti-human stomach adenocarcinoma monoclonal antibody AMC-382 (hereinafter referred to as "AMC-382" for short) is a monoclonal antibody prepared by the present inventors and a serodiagnostic system using the same can detect stomach cancer at a positive rate of 61.5% (European Patent Application No. 87 103 140.7 filed on March 5, 1987). GB-A-2 142 428 teaches a method for use in diagnosing human adenocarcinoma. Polyclonal or monoclonal anti-K- and anti-P k antibodies are employed to detect either the antigenic determinant K which is specific to adenocarcinomas or the antigenic determinant P k associated with determinant K. The determinants K and P k are regarded as part of the tumor marker antigen CEA (carcinoembryonic antigen). Under the present conditions, the above-mentioned serodiagnostic system for lung adenocarcinoma which uses ALC-186 and that for stomach adenocarcinoma which uses AMC-382 are of great clinical value. In cancer detection using sera, however, an improved detection method which could possibly give higher positive rates is desired yet prior to the present invention has not been realized. SUMMARY OF THE INVENTION The present inventors have found that when an enzyme immuno-assay in serodiagnosis of adenocarcinomas is performed by the sandwich technique using as a first antibody a mixture of anti-human lung adenocarcinoma monoclonal antibody ALC-186 and anti-human stomach adenocarcinoma antibody AMC-382 and, as a second antibody, a mixture of ALC-186 and AMC-382 each in an enzyme-labeled form, significantly increased positive rates can be obtained as compared with the systems in which either of the monoclonal antibodies is used by itself. The present invention has been completed based on the above finding. Serodiagnostic test kits are also described. DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, adenocarcinoma antigens in serum samples can be assayed by immobilizing, on a solid phase, a mixture of the anti-human lung adenocarcinoma monoclonal antibody ALC-186 and the anti-human stomach adenocarcinoma monoclonal antibody AMC-382, which serves as a first antibody, allowing a human serum sample to react with the first antibody to bind adenocarcinoma antigens, then allowing a mixture of ALC-186 and AMC-382 each in a labeled form, which serves as a second antibody, to react with the adenocarcinoma antigens bound to the first antibody and determining the quantity of the thus-bound second antibody. According to one preferred embodiment of the invention the second antibody mixture is labeled with an enzyme selected from peroxidase, urease, alkaline phosphatase and beta-galactosidase and the quantity of a bound enzyme-labeled second antibody mixture is measured using an enzyme immunoassay method. According to another preferred embodiment of the invention the second antibody mixture is labeled with a conventional fluorescent label and the quantity of a bound fluorescence-labeled second antibody is measured using a conventional fluorescent antibody method. According to another preferred embodiment of the invention the second antibody mixture is labeled with a conventional immunohistological marker and the quantity of a bound second antibody which is labeled with an immunohistological marker is measured using a conventional immunohistological evaluation method. According to yet another preferred embodiment of the invention the second antibody mixture is labeled with biotin and the quantity of a bound biotinylated second antibody is measured using a biotin-avidin binding assay. The adenocarcinoma antigens to be detected according to the present invention preferably arise from the serum of a patient having an adenocarcinoma selected from lung adenocarcinoma, stomach adenocarcinoma and breast cancer. According to another embodiment of the present invention a serodiagnostic test kit is provided, comprising a first container having a mixture of anti-human lung adenocarcinoma monoclonal antibody ALC-186 and anti-human stomach adenocarcinoma monoclonal antibody AMC-382, in a pharmaceutically acceptable buffer as a first antibody mixture therein, and a second container having a mixture of a labeled anti-human lung adenocarcinoma monoclonal antibody ALC-186 and a labeled anti-human stomach adenocarcinoma monoclonal antibody AMC-382, as a second antibody therein, whereby the first and the second containers are packaged together as a test kit. Preferably the second antibody in the second container is labeled with an enzyme selected from peroxidase, urease, alkaline phosphatase and beta-galactosidase. ALC-186 and AMC-382 are monoclonal antibodies previously disclosed by the present inventors in European Patent Applications No. EP-A-218 257 and No. EP-A-235 817 filed on October 9, 1986 and March 5, 1987. Hybridoma strains capable of producing said monoclonal antibodies, namely the strains ALC-186 and AMC-382 as so referred to herein, can be produced in the manner described below. Both strains have been deposited with the European Collection of Animal Cell Culture in Great Britain under the deposit numbers ECACC 85082903 and ECACC 86022701, respectively, under the Budapest Treaty. Eight- to ten-week old female BALB/c or nude mice treated with pristane® [intraperitoneally administered with 0.5 ml of 2,6,10,14-tetramethylpentadecane (pristane®) and fed for 1- to 2-weeks] are intraperitoneally injected with the cells of the strain ALC-186 or AMC-382 at a dose of 2-4 x 10⁵⁻⁷ cells per animal. In 10 to 21 days, the hybridoma cells produce ascites carcinoma in the mice. The ascitic fluid is collected from such a mouse, subjected to salting out with 5% ammonium sulfate, dialyzed against a solution of 1.83 g/liter of disodium phosphate, 0.21 g/liter of monopotassium phosphate and 7.65 g/liter of sodium chloride (pH 7.2; hereinafter referred to as "PBS" for short) with NaCl added to 0.5 M, and applied to a Cellulofine® GCL-2000 (Seikagaku Kogyo) column (bed volume 750 ml) at a flow rate of 15 ml/hr. An IgM fraction is collected and used as a purified monoclonal antibody. The protein quantity is estimated by the Folin method, followed by calculation based on the absorbance at 280 nm [1.0 (OD₂₈₀) is approximately equal to 1 mg of immunoglobulin M per milliliter]. The monoclonal antibodies thus obtained are evaluated for specificity characteristics based on the reactivities with normal and tumor tissues and membrane components thereof as derived from a variety of human organs obtained from a plurality of subjects, the reactivities with a variety of normal human or tumor cell lines and membrane components derived therefrom, the reactivities with the hitherto known carcinogenic embryonic antigens (e.g., CEA) and the reactivities with normal human-derived and patient-derived sera, among others, as determined by the enzyme immunoassay method, fluorescent antibody method, immunohistological evaluation method (PAP method), etc. Enzyme labeling of the monoclonal antibodies ALC-186 and AMC-382 is performed by the periodic acid crosslinking method [Journal of Histochemistry and Cytochemistry, 22 , 1084-1091 (1971)] using, as the enzyme, peroxidase, urease, alkaline phosphatase or β-galactosidase, for instance. For example, the periodic acid crosslinking using peroxidase may be conducted in the following manner: Peroxidase is dissolved in 1 ml of 0.3 M sodium bicarbonate buffer (pH 8.1), 0.1 ml of a 1% ethanolic solution of fluoro-2,4-dinitrobenzene (DNP) is added, 1 ml of 0.06 M periodic acid is then added and the reaction is allowed to proceed at room temperature for 30 minutes. The reaction mixture is dialyzed against 0.01 M carbonate buffer (pH 9.5) to give a dialyzate containing peroxidase having a DNP-modified aldehyde group. To the dialyzate is added 5 mg of the antibody. After 2 hours of reaction at room temperature the reaction mixture is dialyzed against 0.01 M phosphate buffer (pH 7.2) and the dialyzate is subjected to gel filtration using a Sephadex® G-100 chromatographic column. The active fraction thus obtained is used as the enzyme labeled antibody. When peroxidase is used as the enzyme, an ABTS substrate solution [solution of 550 mg of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt in 1 liter of 0.1 M citrate buffer (pH 4.2) with hydrogen peroxide added to 1 µl/ml just prior to use] is used and the color developed is measured in terms of the absorbance at 415 nm (OD₄₁₅ nm). For urease, a solution containing 8 mg of bromocresol purple, 100 mg of urea and 0.2 mM EDTA per 100 ml thereof and having a pH of 4.8 is used as the substrate and the measurement is performed by the method described in Journal of Immunological Methods, 53 , 187-194 (1982). For alkaline phosphatase, a solution of 1 mg/ml of p-nitrophenyl phosphate in 0.1 M diethanolamine buffer is used as the substrate. The enzyme labeling can also be effected through biotin-avidin bonding. Labeling with biotin can be conducted by the method described in the Journal of Histochemistry and Cytochemistry, 27 , 1131-1139 (1979). The serodiagnosis is performed as follows: A 1:1 mixture of ALC-186 and AMC-382 each in a concentration of 10 to 100 µg/ml is used as the first antibody. The mixture is distributed into the wells of a 96-well plate for EIA (50 to 200 µl per well). The plate is allowed to stand at 4°C to room temperature for 2 hours to two-overnights. After washing with PBS, 200 µl of 1% bovine serum albumin (BSA)-PBS is added to each well. The plate is allowed to stand at 4°C to room temperature for 2 hours to overnight and then washed thoroughly with PBS. Then, 50 to 100 µl of a 1- to 100-fold dilution of a serum sample is added to each well. After allowing the wells to stand at 4°C to room temperature for 2 hours to overnight, the plate is washed thoroughly with PBS. Thereafter, a 1:1 mixture of biotinylated ALC-186 (10 to 100 µg/ml) and biotinylated AMC-382 (10 to 100 µg/ml) is added, as the second antibody, to the wells (50 to 100 µl per well) and the plate is allowed to stand at 4°C to room temperature for 2 hours to overnight. The plate is washed thoroughly with PBS, avidinbiotin-peroxidase (Vector) (10 µg/ml) is added to the wells (50 to 100 µl), the plate is allowed to stand at room temperature for 10 to 30 minutes, and then the reaction is terminated by adding a 5% SDS solution in an amount of 50 to 100 µl per well. The OD₄₁₅ value is measured for each well and the antigen quantity in the serum sample is calculated based on the intensity of the color developed. By comparing the antigen levels in the sera of healthy subjects with those in the sera of cancer patients, a normal level range is defined. When the level in question exceeds this range, the test for adenocarcinoma is regarded as positive. The following non-limiting examples illustrate the invention in further detail. All weights and percent ages are by weight unless otherwise indicated. EXAMPLE 1 A 1:1 mixture of ALC-186 (10 µg/ml) and AMC-382 (10 µg/ml) was distributed as the first antibody into the wells of a 96-well plate for EIA (Flow Laboratories) (100 µl per well). After standing overnight at 4°C, the plate was washed with PBS. Then, 1% BSA-PBS was added (200 µl per well). After standing overnight at 4°C, the plate was washed thoroughly with PBS. Ten-fold dilutions of sera of healthy subject (20 samples), sera of lung cancer patients (23 samples), sera of stomach cancer patients (20 samples), serum of a breast cancer patient (1 sample) and serum of a malignant lymphoma patient (1 sample) were respectively added to the wells in an amount of 50 µl per well. After standing overnight at 4°C, the plate was washed thoroughly with PBS. A 1:1 mixture of biotinylated ALC-186 (10 µg/ml) and biotinylated AMC-382 (10 µg/ml) was distributed, as the second antibody, into the wells (100 µl per well). After standing overnight at 4°C, the plate was washed thoroughly with PBS. Then, avidin-biotin-peroxidase (10 µg/ml) was added to the wells (100 µl per well). After standing at room temperature for 1 hour, the plate was washed thoroughly with PBS. The ABTS substrate solution mentioned hereinabove was distributed in 100 µl portions into the wells. After 30 minutes of reaction at room temperature, the reaction was terminated by adding a 5% SDS solution (100 µl per well). The color developed was measured for each well using an absorptiometer (OD₄₁₅). The results thus obtained are shown in Table 1. In control runs, serodiagnostic assaying was performed with the same serum samples in the same manner as above except that ALC-186 and AMC-382 was used by itself as the first antibody and biotinylated ALC-186 or biotinylated AMC-382 was used alone as the second antibody. The results obtained in these control runs are also shown in Table 1. The cut-off value employed for each assay system was equal to mean OD₄₁₅ + 25D (SD being standard deviation) for 20 healthy subjects used in each assay system. As is evident from Table 1, the system using the 1:1 mixture of ALC-186 and AMC-382 gave significantly increased positive rates in cancer patients, particularly in patients with adenocarcinomas such as lung adenocarcinoma, stomach adenocarcinoma and breast cancer. REFERENCE EXAMPLE 1 Purification of monoclonal antibody ALC-186 Pristane®-treated 8-week-old female BALB/c mice were intraperitoneally injected with the hybridoma strain ALC-186 in a dose of 4 x 10⁶ cells per animal. In 10 to 21 days, the hybridoma produced ascitic carcinoma. The ascitic fluid was collected from ascitic fluid-bearing mice (5 to 10 ml per animal), deprived of solid matter by centrifugation (3,000 rpm, 5 minutes), subjected to salting out with 50% ammonium sulfate and dialyzed against PBS supplemented with 0.5 M NaCl. The dialyzate was applied to a Cellulofine® GCL-2000 (Seikagaku Kogyo) column (bed volume 750 ml) at a flow rate of 15 ml/hr. An IgM fraction was collected and used as the purified monoclonal antibody. REFERENCE EXAMPLE 2 Purification of monoclonal antibody AMC-382 Pristane®-treated 8-week-old female C57BL/6 mice were intraperitoneally injected with the hybridoma strain AMC-382 in a dose of 4 x 10⁶ cells per animal. In 10 to 21 days, the hybridoma produced ascitic carcinoma. The ascitic fluid was collected from ascitic fluid-bearing mice (5 to 10 ml per animal), deprived of solid matter by centrifugation (3,000 rpm, 5 minutes), subjected to salting out with 50% ammonium sulfate and dialyzed against PBS supplemented with 0.5 M NaCl. The dialyzate was passed through a Cellulofine® GCL-2000 (Seikagaku Kogyo) column (bed volume 750 ml) at a flow rate of 15 ml/hr. An IgM fraction was collected and used as purified antibody. The present invention thus makes it possible to perform serodiagnosis of human adenocarcinomas in a simple, efficient and reliable manner.

Claims: A serodiagnostic method for assaying adenocarcinoma antigens in a serum sample which comprises the steps of: (1) immobilizing, on a solid base, a first antibody which is a mixture of anti-human lung adenocarcinoma monoclonal antibody ALC-186 obtainable from a hybridoma strain deposited under the number ECACC 85082903 and anti-human stomach adenocarcinoma monoclonal antibody AMC-382 obtainable from a hybridoma strain deposited under the number ECACC 86022701; (2) allowing a human serum sample to react with said first antibody to bind adenocarcinoma antigens; (3) allowing a second antibody, which is a mixture of anti-human lung adenocarcinoma monoclonal antibody ALC-186 and anti-human stomach adenocarcinoma monoclonal antibody AMC-382, each antibody in a labeled form, to react with said adenocarcinoma antigens; and (4) determining the quantity of the thus-bound second antibody. The serodiagnostic method of claim 1 in which the second antibody of step (3) is labeled with an enzyme selected from peroxidase, urease, alkaline phosphatase and beta-galactosidase. The serodiagnostic method of claim 1 or 2 in which the quantity of a bound enzyme-labeled second antibody is measured using an enzyme immunoassay method. The serodiagnostic method of claim 1 in which the quantity of a bound fluorescence-labeled second antibody is measured using a fluorescent antibody method. The serodiagnostic method of claim 1 in which the quantity of a bound second antibody which is labeled with an immunohistological marker is measured using an immunohistological evaluation method. The serodiagnostic method of claim 1 in which the quantity of a bound biotinylated second antibody is measured using a biotinavidin binding assay. The serodiagnostic method of claim 1 in which the adenocarcinoma antigens to be detected arise from the serum of a patient having an adenocarcinoma selected from lung adenocarcinoma, stomach adenocarcinoma and breast cancer. A serodiagnostic test kit comprising: (a) a first container having a mixture of anti-human lung adenocarcinoma monoclonal antibody ALC-186 and anti-human stomach adenocarcinoma monoclonal antibody AMC-382, in a pharmaceutically acceptable buffer as a first antibody mixture therein; (b) a second container having a mixture of a labeled anti-human lung adenocarcinoma monoclonal antibody ALC-186 and a labeled anti-human stomach adenocarcinoma monoclonal antibody AMG-382, as a second antibody therein; the first and the second containers packaged together as a test kit. The serodiagnostic test kit of claim 8 in which the second antibody in the second container is labeled with an enzyme selected from peroxidase, urease, alkaline phosphatase and beta-galactosidase.

Inventors: HANAI, NOBUO, KUMAGAI, AKIKO, SHITARA, KENYA, YOSHIDA, HAJIME
Applicants: KYOWA HAKKO KOGYO CO., LTD.
Application year: 1987
Main classification: G01N33
IPC codes: G01N33, C12Q1