JPS60363A - Novel method for quantitative determination of antigen - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying a novel antigen.

In recent years, in the field of medicine, it has become an extremely important issue to easily and quickly quantify antigens with high reliability for purposes such as disease diagnosis.

Conventional methods for quantifying antigens using immunochemical JILJ standard methods include radioimmunoassay (radioimmunoassay) (A), enzyme immunoassay (B), reverse passive hemagglutination method (C), and radial immunodiffusion method), etc., but each of these quantitative methods had the following drawbacks.

Method A: Requires a washing operation, and requires special equipment due to the use of radioisotopes, resulting in huge equipment costs and complicated procedures.

Method B generally requires a cleaning operation and takes a long time to make a determination.

Method C: Requires a stepwise dilution of the sample to 2 times with a dilimator and an operation of dropping the diluent etc. with a dropper, which is complicated. In addition, it takes a long time to make the determination, and since the antigen amount is determined based on the final value of two-fold serial dilution, the measurement may be complicated.

Method D: It takes a long time to make a determination and is insufficient in sensitivity.

The present invention has been made as a result of intensive research aimed at improving these drawbacks, and has resulted in the discovery of a method for quantifying antigens with high sensitivity, simply and quickly. That is, the method of the present invention uses different glazes.
After adding an antigen to a red blood cell suspension to which one type of antibody has been bound, hemolysis of red blood cells occurs when the other antibody and complement are added and reacted. Intrared blood cell components such as cyhemoclobin become liberated. This is a method for indirectly quantifying antigens by measuring components in red blood cells such as hemoglobin.

In the method of the present invention, an antigen is added to a red blood cell suspension to which one antibody has been bound, and it is thought that an antigen-antibody reaction occurs at this stage, and the addition of complement causes hemolysis due to a complement fixation reaction. However, no hemolysis occurred. However, it was discovered that hemolysis due to complement fixation reaction occurs only when the other antibody is further reacted with the antibody.

Examples of antigens that can be quantified in the method of the present invention include α-fetoprotein, T3°T4 r HBs
, immunoglobulin G, immunoglobulin M, myoglobin, etc., but it is not limited to these antigens; it is capable of quantifying all types of antigens that contribute one complement to the antigen-antibody reaction. It is something.

Furthermore, when binding antibodies to red blood cells in the method of the present invention, it is preferable to chemically adsorb the antibodies to the red blood cells. Many methods have already been proposed for carrying antibodies on red blood cells. For example, there is a method using tannin rL chromium chloride, water-soluble carbodiimide, and the like.

Furthermore, the red blood cells used in the method of the present invention are not limited, and any type of red blood cells may be used. However, in order to increase the sensitivity of quantitative determination, red blood cells that are susceptible to hemolysis due to complement fixation reactions, such as sheep red blood cells, are preferred.

Furthermore, in the method of the present invention, the type of complement is not particularly limited, and complements with high complement activity are desirable. For example, guinea pig complement.

The following describes how the method of the present invention differs from conventional complement fixation reaction tests. The complement fixation reaction test is a method conventionally known as an antigen quantification method or an antibody quantification method. That is, when an antibody binds to an antigen, molecular changes occur in the antibody molecule, complement binds to the antibody, and the antibody is activated and consumed.

This is called the complement fixation reaction. Using this reaction, the strength of the antigen-antibody reaction can be indirectly determined from the amount of complement consumed. For example, if the amount of antigen is kept constant, the amount of antibody can be measured, and if the amount of antibody is kept constant, the amount of antigen can be measured.The consumption of complement due to the antigen-antibody reaction is due to the amount of sheep red blood cells bound to sheep red blood cells. The hemolysis of sheep red blood cells is used as an indicator.In the conventional complement fixation reaction test, antigens or antibodies are determined from hemolysis of sheep red blood cells bound to antibodies against sheep red blood cells due to residual complement consumed in such antigen-antibody reactions. However, in the method of the present invention, the antigen to be quantified is directly reacted with the antibody on the surface of red blood cells, and the antigen is indirectly quantified from the hemolysis of the red blood cells due to the complement fixation reaction.

In conventional complement fixation reaction tests, the hemolysis of sheep red blood cells bound to antibodies against sheep red blood cells due to residual complement consumed in the antigen-antibody reaction is used as an indicator, so the amount of complement must be constant, and the amount of complement must be constant. 5 to eliminate the influence of existing complement in serum
Immobilization must be carried out for 30 minutes at 6°C. Therefore, it could not be used for quantifying heat-labile antigens. However, in the method of the present invention, it is sufficient that the complement level is above a certain level, and there is no influence of existing complement in the test serum, no immobilization is required, and even heat-labile antigens can be quantified. Furthermore, conventional complement fixation reaction tests require complicated operations such as diluting the test serum in 2-fold steps and require a long time to make a determination, but the method of the present invention requires complicated operations such as 2-fold stepwise dilution, etc. Antigens can be quantified simply and quickly without worrying about operation.

Next, examples of the present invention will be described.

Example (1) For the determination of human α-fetoprotein, 1 volume of sheep erythrocyte sediment washed with physiological saline was added with chloride (0.4
m9/'n') and 2 volumes of anti-human α-7 etoglotein goat antibody (Tan/f 1400 μg/rnl) were mixed and reacted at room temperature for 30 minutes, then washed with physiological saline. % suspension. To 50 μt of this antibody-sensitized sheep red blood cell suspension, 50 μt of a sample containing various concentrations of human α-fetoprotein and 50 μt of anti-human α-fetoprotein rabbit antibody were added, and further 10 μt of the antibody-sensitized sheep red blood cell suspension was added with Peronal buffer.
Guinea pig complement diluted 0x3. Add Qmr and 37℃
After reacting for 30 minutes, low-speed centrifugation was performed and the absorbance (416 nm) of hemoglobin in the supernatant was measured.

The results are as shown in Figure 19. Based on the linear relationship between the acceptability of human α-fetoprotein and the absorbance, human α-fetoprotein
Fetoprotein can be quantified.

Example (2) In the determination of human immunoglobulin G, chromium chloride (0.4m9/
ml) 2 volumes and anti-human immunoglobulin G (γ-chain specific) sheep antibody (tan I? amount 400 μg/ml)
Two volumes were mixed, reacted for 30 minutes at room temperature, and then washed with physiological saline to obtain a suspension of 8 cells. To 50 μt of this antibody-sensitized sheep red blood cell suspension, 50 μt of a sample containing various concentrations of human immunoglobulin G and anti-human immunoglobulin G (γ
-chain t1\isomer) guinea pig complement 3.50 μt of rabbit antibody was added and further diluted 100 times with peronal buffer. After adding Oml, low-speed centrifugation was performed to measure the absorbance (416 nm) of hemoglobin in the upper 71 offspring.

The results are shown in FIG. 2. Human immunoglobulin G can be quantified based on the white line relationship between the concentration of human immunoglobulin G and absorbance.

As detailed above, the method of the present invention has remarkable effects such as excellent sensitivity and ability to quantify antigens simply and quickly.

An explanatory diagram of the relationship between the concentration and absorbance of human α-fetoprotein, and FIG. 2 shows the relationship between the concentration and absorbance of human α-fetoprotein.

FIG. 2 is an explanatory diagram showing the relationship between the absorbance and absorbance of human immunoglobulin G in the method.

Claims (1)

[Claims] After removing the antigen from a red blood cell suspension bound to one of two types of antibodies from different species, red blood cells are made by reacting with the other antibody and complement. A novel antigen quantification method characterized by causing hemolysis and indirectly quantifying the antigen from the hemolysis phenomenon.