JPH04249770A - Cancer diagnostic agent - Google Patents

Abstract

PURPOSE:To discover the presence of cancer at a relatively early stage by measuring the autoantibody to a carcinoembryonic antigen (CEA). CONSTITUTION:An autoantibody appears in early cancer with respect to CEA being one of tumor markers. Therefore, the early diagnosis of cancer can be performed by the efficient and accurate measurement of an anti-CEA autoantibody. With respect to a cancer diagnostic agent, all of chemical agents capable of measuring the anti-CEA autoantibody are contained and all of techniques used in high sensitivity immunoassay are applied and, in these techniques, a sandwich method, a second antibody method, a competition method, a sedimentation method and the like are used. As the tracer used in these methods, enzyme, an isotope, a fluorescent substance, a light emitting substance and other labelling agent are used. As a solid phase for immobilizing CEA, polystyrene beads, plate, latex particles, magnetic fine particles and the like are used and, as an immobilizing method, a physical adsorption method, a covalent bond method, a biotin-avidin bonding method and an antigen-antibody reaction method are used.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention relates to a novel diagnostic agent for cancer, and in particular, it is capable of detecting early cancer, and is expected to make a significant contribution to the field of medical testing and diagnosis. It is.

0002

BACKGROUND OF THE INVENTION Conventionally, serological diagnosis of cancer has mainly been made by measuring substances collectively called tumor markers in the blood. As a tumor marker, carcinoembryonic antigen (Carcinoembryonic antigen)
n, CEA), α-fetoprotein (AFP), CA
19-9, prostatic acid phosphatase, etc. are known. Although the positive rate of these tumor markers differs depending on the type of cancer, the blood concentration of these tumor markers generally increases as the tumor grows. Therefore, it has been used for monitoring the effects of cancer treatment and diagnosing prognosis. However, these tumor markers are detected in the blood only when the cancer has grown considerably, and it has been completely difficult to detect these tumor markers in the early stages of cancer development, so-called early cancer. In other words, it was not useful for screening for early stage cancers, which have an extremely high chance of being cured.

For example, the currently known sandwich method C
In the EA measurement kit, CEA 0.5ng/ml to 1
It is possible to measure only 000 ng/ml (Nippon Rinsho, 34 [6] (1976), p1274-1279).
), it was not possible to measure extremely low concentrations of CEA, and it was not possible to detect early cancer.

0004

SUMMARY OF THE INVENTION An object of the present invention is to develop a new system that is useful for truly effective serological diagnosis of cancer, which leads to early detection of cancer.

[0005]

[Means to solve the problem] In view of the current situation,
As a result of intensive research to develop a new serological diagnostic system that can detect early cancer, the present inventors discovered that autoantibodies against CEA, one of the tumor markers, appear in early cancer. Furthermore, we have established a method for efficiently and accurately measuring the anti-CEA autoantibody, and have now completed the present invention.

[0006] That is, the basic technical idea of the present invention is to perform early diagnosis of cancer by measuring autoantibodies against CEA. The present invention relates to reagents for cancer diagnosis, that is, cancer diagnostic agents. The present invention will be explained in detail below.

[0007] The cancer diagnostic agent according to the present invention includes all drugs (systems) capable of measuring anti-CEA autoantibodies, but autoantibodies to CEA can be measured by enzyme immunoassay (EIA), radio All the techniques used in existing highly sensitive immunoassays such as immunoassay (RIA), fluorescence immunoassay (FIA), and luminescence immunoassay can be applied, and therefore all the drugs (systems) used in these assays are applicable to the present invention. It can be used as a cancer diagnostic agent.

[0008] Examples of methods for measuring anti-CEA autoantibodies include the following method.

[Sandwich method] Solid phase-immobilized CEA is reacted with anti-CEA autoantibody in the sample, and the complex formed on the solid phase is further reacted with tracer-labeled CEA, or biotin-bound CEA and avidin-bound The tracers can be reacted simultaneously or staggered and solid-phase CEA-
A method of forming an autoantibody-tracer-labeled CEA sandwich complex, measuring the tracer on a solid phase, and measuring anti-CEA autoantibodies.

[Second antibody method] CEA immobilized on a solid phase is reacted with anti-CEA autoantibody in the sample, and a tracer-labeled anti-human immunoglobulin antibody (labeled second antibody) is added to the complex formed on the solid phase. A method of reacting to form a CEA-autoantibody-second antibody complex on a solid phase, measuring a tracer on the solid phase, and measuring anti-CEA autoantibodies.

[Competitive method] Anti-CEA autoantibodies in the sample and tracer-labeled anti-CEA antibodies are competitively reacted with solid phase-immobilized CEA, and the tracer on the solid phase is measured.
A. Method for measuring autoantibodies.

[Precipitation method] After reacting the tracer-labeled CEA with the anti-CEA autoantibody in the sample, the resulting complex is precipitated using polyethylene glycol, and the tracer in the precipitate or supernatant is measured, Method for measuring anti-CEA autoantibodies.

[0013] In each of the above-mentioned methods, tracers include enzymes (peroxidase, β-galactosidase, glucose oxidase, alkaline phosphatase,
G-6-P dehydrogenase, etc.), isotope (12
5I, 131I, etc.), fluorescent substances (fluorescein, FI
TC, rhodamine, etc.), luminescent substances (ferritin, luciferin, luminol, etc.), and other commonly used labeling agents can all be freely used.

As the solid phase for immobilizing CEA, conventionally used polystyrene beads, plates, latex particles, magnetic particles, etc. can be used, and there are no restrictions on the material or shape. All known methods for immobilization can be used, including physical adsorption, covalent bonding, biotin-avidin binding, and antigen-antibody reaction. Further, for the preparation of tracer-labeled CEA, tracer-labeled antibodies, etc., existing techniques such as chemical bonding methods using amino groups or thiol groups and labeling methods using biotin-avidin bonding can be used as appropriate.

In the sandwich method, CEA immobilized on a solid phase and the sample and tracer-labeled CEA are incubated simultaneously or at different times. The reaction time depends on the solid phase used and the type of tracer, but it usually takes several minutes to several hours. Further, the tracer may be directly labeled with CEA, but if the tracer is an enzyme, biotin-CEA labeled with biotin, which is easy to label, can also be used. In this case, solid phase-immobilized CEA, specimen, biotin-bound CEA, and avidin-bound tracer (enzyme) are incubated simultaneously or at staggered times. When changing the time, it is preferable to perform a so-called BF separation operation to remove unreacted substances during the reaction. Finally, a solid phase CEA-autoantibody-biotinylated CEA-avidin-bound tracer (enzyme) complex is obtained. The method for measuring tracers varies depending on the type, but existing methods can be used, and the degree of activity and intensity is proportional to the amount of autoantibodies in the sample, so by simultaneously measuring an appropriate control sample or standard solution. , autoantibodies can be measured. This method allows autoantibodies to be measured with relatively high sensitivity and is the most preferred method.

In the second antibody method, a sample is incubated with CEA immobilized on a solid phase, and after BF separation by washing, a tracer-labeled anti-human immunoglobulin antibody is reacted. The anti-human immunoglobulin antibodies used in this case are polyclonal antibodies derived from various animals or monoclonal antibodies from mice and other animals. The reaction time and tracer measurement method are the same as the sandwich method.

In the competitive method, CEA immobilized on a solid phase, a specimen, and a tracer-labeled anti-CEA antibody are incubated at the same time or at different times to react. The tracer-labeled antibody reacts with the immobilized CEA competitively with the autoantibody in the sample, and the amount bound to the solid phase is inversely proportional to the amount of autoantibody in the sample. The antibody used for tracer labeling is preferably an animal-derived polyclonal antibody.

In the precipitation method, a sample and tracer-labeled CEA are incubated to generate an autoantibody-tracer-labeled CEA complex, and then polyethylene glycol (PEG) or other known precipitating agent is added to the reaction system to separate the resulting complex. precipitate. The supernatant and precipitate are centrifuged, and the tracer in the precipitate or supernatant is measured. The amount of tracer in the precipitate is proportional to the autoantibody in the sample.

The diagnostic agent according to the present invention can be easily prepared by dispensing each drug appropriately, preparing plates and test tubes, and further preparing a buffer solution and an enzyme substrate solution according to the conventional method for preparing an immunoassay kit. Or, in the case of preparation at the time of use, additionally prepare water (buffer, physiological saline, etc.) for diluting or dissolving the lyophilized prescribed drug, and further prepare control serum (plasma), etc., and prepare as desired. It is particularly useful in practice if you prepare it as a kit.

For example, the contents of a diagnostic kit using the sandwich method include a test tube containing CEA-immobilized polystyrene balls, a phosphate buffer, a serum diluent, a tracer-labeled CEA solution, an enzyme conjugate, an enzyme substrate, etc. These may be appropriately packaged and sold as a kit.

[0021]

[Example] Examples and reference examples of measuring anti-CEA autoantibodies in serum, plasma, and other various specimens taken outside the body for the purpose of cancer diagnosis using the diagnostic agent according to the present invention will be described below. However, these examples are intended to explain the present invention in more detail, and the present invention is not limited to these examples.

[0022]

[Example 1 Anti-CEA autoantibody measurement by sandwich method] 25 μl of sample, phosphate buffer (pH 7.3, 0.5 μl)
(contains 1M NaCl, 0.5% bovine serum albumin)
200 μl, CEA-immobilized polystyrene balls (PSB
) was incubated in a test tube at 37°C for 2 hours. After washing the PSB three times with 3 ml of distilled water,
200 μl of biotin-labeled CEA solution was added and reacted at 37° C. for 1 hour. After washing three times with distilled water, 200 μl of avidin-conjugated peroxidase was added, the reaction was further carried out at 37° C. for 1 hr, and the cells were washed in the same manner as before. 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (AB
Add 500 μl of a coloring solution consisting of TS) and hydrogen peroxide,
After carrying out the enzymatic reaction for 1 hour at room temperature, 1% oxalic acid was added to stop the reaction. Measure the absorbance at 420 nm,
Anti-CEA autoantibodies in the specimens were measured. The results are shown in Table 1.

[0023]

[Table 1]

[0024]

[Example 2 Anti-CEA autoantibody measurement by second antibody method] 25 μl of the sample and 1 piece of CEA-immobilized PSB were heated at 37°C.
The reaction was carried out for 2 hours, and washing was performed three times using 3 ml of distilled water. 200 μl of peroxidase-labeled rabbit anti-human immunoglobulin antibody solution was added, and the mixture was reacted at 37° C. for 1 hour, and then washed in the same manner. An enzymatic reaction was performed in the same manner as in Example 1, and the absorbance was measured to determine the autoantibody in the sample. The results are shown in Table 1.

[0025]

[Example 3 Anti-CEA autoantibody measurement method using competitive method]
100 μl of appropriately diluted specimen, 300 μl of peroxidase-labeled rabbit anti-CEA antibody, and CEA-immobilized PSB
One sample was incubated at 37°C for 2 hours, and after washing, the anti-CEA autoantibody in the sample was measured in the same manner as in Example 1. The results are shown in Table 1.

[0026]

[Example 4 Anti-CEA autoantibody measurement by precipitation method] 50 μl of specimen, 50 μl of 125I-labeled CEA solution, and 0.5 μl of 125I-labeled CEA solution.
Add 150 μl of 01M ammonium acetate (pH 6.8) to 4
℃, overnight incubation. Add 250 μl of 25% polyethylene glycol, centrifuge at 3000 rpm for 20 minutes, and calculate the radioactivity ratio of the precipitate to the total radioactivity (%
) was measured. The results are shown in Table 1.

[0027]

[Reference Example 1 Measurement of CEA by sandwich EIA method] 50 μl of sample, peroxidase-labeled rabbit anti-CE
300 μl of antibody solution A and one PSB immobilized with anti-CEA monoclonal antibody were incubated at 37° C. for 2 hours, and washed three times with 2 ml of distilled water. A coloring solution was added in the same manner as in Example 1, an enzyme reaction was performed, and the absorbance was measured. The CEA concentration of the sample was read from the standard CEA calibration curve. The results are shown in Table 1.

As is clear from the results shown in Table 1, the cancer diagnostic method using the present invention is effective against the conventionally widely used CE.
It was confirmed that early gastric cancer cases with normal A concentration can be detected with high sensitivity.

[0029]

[Effects of the Invention] Conventional tumor markers can only be detected in the blood of cancer patients at a fairly advanced stage, and are powerless in detecting early cancer. However, according to the present invention, CEA in blood
By measuring autoantibodies against cancer, it has become possible to detect the presence of cancer at a relatively early stage. It is extremely significant that the present invention enables the detection of early cancer, which is difficult to detect using conventional physiological tests and serological tests.

Claims (7)

[Claims] 1. A cancer diagnostic agent comprising a drug for measuring autoantibodies against carcinoembryonic antigen (CEA). 2. The drug consists of immobilized CEA and tracer-labeled CEA, and anti-CEA is prepared according to the sandwich method.
2. The cancer diagnostic agent according to claim 1, which measures EA autoantibodies. 3. The drug consists of immobilized CEA and a tracer-labeled anti-CEA antibody, and the anti-CEA
2. The cancer diagnostic agent according to claim 1, which measures A autoantibody. 4. The cancer diagnostic agent according to claim 2, which uses biotin-bound CEA and an avidin-bound tracer instead of tracer-labeled CEA. 5. The cancer diagnostic agent according to claim 1, wherein the agent comprises immobilized CEA and a tracer-labeled anti-human immunoglobulin antibody, and is for measuring anti-CEA autoantibodies according to a second antibody method. . 6. The cancer diagnostic agent according to claim 1, wherein the agent comprises tracer-labeled CEA and a precipitant, and is used to measure anti-CEA autoantibodies according to a precipitation method. 7. The cancer diagnostic agent according to claim 1, wherein the tracer is selected from enzymes, isotopes, fluorescent substances, and luminescent substances.