JPH0331227B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for measuring agglutination reactions using insoluble carrier particles. In recent years, in the field of clinical testing, serological diagnosis of certain diseases has become important. For this diagnosis, accurate, simple, and rapid quantification of antigens and antibodies has become an extremely important issue. The present invention aims to solve this problem. Generally, an antigen-antibody reaction occurs within a certain concentration range to form a complex. If this concentration ratio is extremely biased, it becomes difficult to form an antigen-antibody complex. When the amount of antigen is increased in the presence of a certain amount of antibody, the amount of precipitate due to complex formation increases in proportion to the antigen concentration up to a certain amount of antigen, but once the antigen concentration exceeds a certain point, the opposite occurs. The amount of sediment decreases. This decrease is well known as a solubilization phenomenon of the complex due to antigen excess. This can also occur in antigen measurement reactions using serological latex reagents. Serological latex reagents are usually made by sensitizing latex particles with a particle size of 0.005 to 1 μm with antibodies or antigens and dispersing them in a weakly alkaline buffer. However, since there is a limit to the amount of antibody or antigen that can be sensitized to a certain amount of latex particles, there is also a limit to the amount of antigen or antibody that can be measured. Therefore, when measuring high-concentration antigens, the above-mentioned antigen excess phenomenon may result in erroneously measured low values.
In this case, attempts are made to avoid this error by diluting the specimen (sample), but this is complicated. For example, when measuring immunoglobulin G (IgG), it is customary to dilute the sample serum approximately 1,000 to 10,000 times.
This is a major drawback when considering the need to simplify the measurement operation of clinical test drugs. Conventionally, a method has been proposed in which an antigen-antibody agglutination reaction is performed using a reagent in which insoluble carrier particles such as latex are sensitized with antibodies or antigens, and the turbidity of the complex formed as a result of the reaction is optically measured. [Unexamined Japanese Patent Publication No. 54-108693, Unexamined Japanese Patent Application No. 55-159157, Unexamined Unexamined Publication
57-1970 and Sawai and Ishida, Inspection and Technology: 10(6),
555 (1982) etc.]. However, even with these methods, when the sample concentration is high, extensive dilution is required without exception, resulting in large errors due to dilution and erroneous measurement values due to antigen excess phenomenon caused by the narrow measurement range. There is also a risk of releasing. As a result of studying the above, the present inventor found that when measuring a high concentration antigen such as immunoglobulin present in a sample serum, insoluble carrier particles are sensitized with free antibodies that can specifically react with the antigen in the sample. By adding the antibody to the reaction system together with the antibody, the measurement range is expanded and the frequency of antigen excess phenomenon is avoided, and the dilution of the sample serum is minimized, thereby creating an accurate and simple method for measuring high-concentration antigens. This finding led to the completion of the present invention. That is, the present invention provides a method in which insoluble carrier particles are sensitized with an antibody, the antibody is reacted with a sample, and the aggregation of this reaction mixture is optically measured. This is an agglutination reaction measurement method characterized by adding a free antibody capable of specifically reacting with an antigen in a specimen to a reaction system to perform an antigen-antibody reaction. According to the method of the present invention, the agglutination reaction is performed by sequentially sensitizing insoluble carrier particles with a free monospecific antibody capable of reacting with the antigen in the antibody sample serum and the normal serum sample serum of an antibody-producing animal in a liquid medium. The reaction is caused by shaking and mixing for a minute, and after dilution to a constant volume with a diluent, the agglutination based on the antigen-antibody reaction is irradiated with light, and it can be quantitatively measured by the change in absorbance at that time. Of course, the order of the pipetting operations described above is arbitrary and there is no need to adhere to the above numerical order, but it is preferable to react only the sample serum last. The insoluble carrier particles used in the present invention are fine particles of organic polymeric substances, such as polystyrene, carboxy-modified, sulfonic acid-modified, latex obtained by emulsion polymerization such as styrene-acrylonitrile copolymers, and individually dispersed staphylococci. ,
Coccus type bacteria such as streptococcus, inorganic oxides such as silica, silica-alumina, alumina, other mineral powders, metals, etc. are used. There are many known methods for sensitizing antibodies to such insoluble carrier particles, and any of these methods may be used. Also,
A buffer solution such as physiological saline, phosphoric acid, or Tris-HCl is used as the diluent. The target that can be measured by the method of the present invention is
Immunoglobulins such as IgG, IgA, IgM, etc., albumin, ceruloplasmin, transferrin,
Examples include antitrypsin, and it is particularly effective for measuring substances such as immunoglobulins that are present in high concentrations in sample serum. In the method of the present invention, when a free antibody is added to an antibody sensitized to an insoluble carrier, nonspecific aggregation may occur due to a lot of antibodies. In such cases, the normal serum of the animal used for counterproduction is added to the reaction system to avoid this nonspecific agglutination. According to the present invention, the amount of antigen that can be measured can be increased and accuracy can also be expected. Accordingly, the dilution factor of the sample serum can be significantly reduced, and the measurement range can be significantly expanded. EXAMPLES Hereinafter, the present invention will be further explained with reference to Examples, but the present invention is not limited thereto. Example 1 (1) Preparation of anti-human IgG antibody A human IgG monospecific antibody produced in a domestic rabbit is used. (The solution was passed through a Sepharose 4B column immobilized with the antigen human IgG chain and purified by Affinitei chromatography.) (2) Preparation of anti-human IgG antibody sensitization latex reagent Latex (manufactured by Nippon Gosei Rubber Co., Ltd., carboxy Denatured type, particle size 0.305μ) was suspended (0.5%) in a pH8 glycine buffer, and 1 volume of this suspension was mixed with anti-human IgG adjusted to 500μg/ml with a phosphate buffer.
Mix with 1 volume of antibody solution, keep at room temperature for 30 minutes, centrifuge at 10,000 x G for 20 minutes to remove rabbit r-globulin molecules adsorbed on the latex particles, and then remove the precipitated latex particles. 1 again
A sensitized latex is prepared by dispersing it in a glycine buffer containing 0.5% BSA. (3) Measurement of latex agglutination reaction Add 20μ of the above anti-IgG latex reagent, 20μ of anti-IgG antibody solution, 20μ of normal rabbit serum diluted 3 times with phosphate buffer, and 20μ of sample serum or standard product into a small test tube and wait for 10 minutes. After shaking and mixing, 4 ml of the diluent was added, and the absorbance at 340 mm was measured using a Hitachi Model 200-10 spectrophotometer, and the aggregation activity was determined by the change in absorbance (-Δ340) relative to the blank. (The concentration is calculated using -△340 of the standard product as a calibration.) Comparative Example 1 () In the measurement of latex agglutination reaction in (3) of Example 1, the effect of free antibody on preventing nonspecific agglutination was evaluated using normal rabbits. Instead of measuring without serum, measure using phosphate buffer (100mM, PH7.5). After carrying out an agglutination reaction according to a predetermined method, the presence or absence of agglutination was determined with the naked eye.
The results are shown in Table 1.

[Table] () The non-specific agglutination prevention effect of human serum components is measured using phosphate buffer (100mM, PH7.5) instead of excluding normal rabbit serum. Aggregation was determined according to (1). The results are shown in Table 2. The latex reagent used was one sensitized with anti-α-fetoprotein antibody (rabbit). The preparation method is based on Example 1 (2).

[Table] −: No aggregation observed with the naked eye +: Appearances with obvious aggregation observed with the naked eye

[Brief explanation of the drawing]

FIG. 1 is a diagram comparing the absorption spectrum obtained when an antigen is added to a latex reagent and aggregated with that of a control (no antigen added).

Claims (1)

[Claims] 1 In a method in which insoluble carrier particles are sensitized with an antibody, the antibody is reacted with a specimen, and the agglutination of this reaction mixture is optically measured, the antigen in the specimen is separated from the antibody sensitized to the insoluble carrier particles. 1. A method for measuring an agglutination reaction using insoluble carrier particles, characterized in that an antigen-antibody reaction is carried out by adding a free antibody capable of specifically reacting to a reaction system.