JPH02120664A - Immunological detection of antigen - Google Patents

Abstract

PURPOSE:To detect an antigen extremely high in specificity by a simple means by utilizing that agglutination reaction is generated together using monoclonal antibody sensitized particles and a polyclonal antibody. CONSTITUTION:Particles sensitized with the monoclonal antibody to an antigen and the polyclonal antibody to said antigen are brought into contact with the antigen and the antigen is detected on the basis of generated agglutination reaction. As the antigen to be detected, any one having an antigen is used and there is no special limit and a hapten having no immunogenecity is also contained. The contact of the polyclonal antibody and the antigen is usually performed after or simultaneously with the contact of monoclonal antibody sensitized particles and the antigen. When the contact of the polyclonal antibody and the antigen is performed before the contact of the monoclonal antibody sensitized particles and the antigen, the antigen can be bonded to the monoclonal antibody sensitized particles and no agglutination is generated according to circumstances.

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] (Industrial Field of Application) The present invention relates to a method for detecting an antigen by immunological agglutination reaction.

(Prior art and problems to be solved by the invention) Conventionally,
As a method for detecting antigens by agglutination reaction, reverse passive agglutination reactions such as reverse passive hemagglutination reactions are widely used and are widely used in clinical tests, but the antibodies used for sensitization are It is an immune serum that is a polyclonal antibody.

For this reason, highly specific antibodies are required, and moreover, labor-intensive specific purification using a large amount of antigen is essential.

If a monoclonal antibody could be used as the sensitizing antibody used in the above-mentioned reverse passive agglutination reaction, the antigen-antibody reaction would be based on a single antigenic determinant, resulting in much higher specificity than immune serum. Since cloned antibodies have no agglutination properties except in exceptional cases, they could not be applied to reverse passive agglutination reactions.

The present inventor discovered that an agglutination reaction occurs when monoclonal antibody-sensitized particles and polyclonal antibodies are used together, and by utilizing this, succeeded in developing an extremely specific antigen detection method. , we have completed the present invention.

[Structure 1 of the Invention (Means and Effects for Solving the Problems) The immunological antigen detection method of the present invention comprises an antigen, particles sensitized with a monoclonal antibody against the antigen, and polyclonal antibodies against the antigen. The feature is that the antigen is detected by the agglutination reaction that occurs upon contact with the antigen.

In the present invention, the antigen to be detected is not particularly limited as long as it has antigenicity, and includes habten that does not have immunogenicity.

Such antigens include, for example, plasma proteins such as ferritin, apoliboprotein, and C-reactive protein; tumor markers such as carcinoembryonic antigen and a-fetoprotein; hormones such as insulin, erythroboetin, and gonadotropin; Mycobacterium tuberculosis, and streptococci. Bacteria and their bacterial body components and produced substances: Viruses such as rotavirus, adenovirus, HB virus, human immunodeficiency virus (HIV) and their constituent proteins:
Examples include microorganisms such as mycoplasma, leptospirosis, and fungi, and their constituent components.

i? Particles that sensitize the monoclonal antibody described in i.
Red blood cells, bacterial cells, latex such as polystyrene,
Particles made of glass, kaolin, silica, etc., particles made of protein, charcoal, etc., as long as they can bind monoclonal antibodies directly or indirectly, can have any composition, but particles with a diameter of 0 It is desirable to have a spherical shape with a value of about .01=lOum, and to be stable for a long period of time. Monoclonal antibodies sensitized to these particles can be obtained by well-known conventional methods.

For example, B cells from antigen-immunized mouse spleen and mouse myeloma cells are fused using polyethylene glycol, etc.
A monoclonal antibody-producing hybridoma may be produced, cultured, or inoculated into mice to proliferate, and then collected from the ascites.

The monoclonal antibody can then be sensitized to the particles using well known methods. For example, when sensitizing polystyrene latex, the latex may be suspended in physiological saline, mixed with a monoclonal antibody, and slowly stirred at room temperature. After sensitization, unadsorbed antibodies are removed by washing, and then the unadsorbed portions of antibodies are blocked with a blocking agent such as albumin, and then suspended in an appropriate solution.

Furthermore, polyclonal antibodies can be prepared by immunizing animals using well-known methods. In this case, antibodies obtained by immunization with antigens containing impurities may be used.

In the present invention, the contact between the polyclonal antibody and the antigen is usually carried out after or simultaneously with the contact between the monoclonal antibody-sensitized particles and the antigen, and before the contact between the monoclonal antibody-sensitized particles and the antigen. When contacting a polyclonal antibody with an antigen, the antigen may not be able to bind to the monoclonal sensitized particles and agglutination may not occur.

Types of agglutination reactions for detecting antigens include bottom agglutination reactions in test tubes or microtiter plates, slide agglutination reactions, and in vitro agglutination reactions.

Among these, for in vitro agglutination reactions, it is easy to obtain quantitative values by measuring the strength of agglutination with a turbidity meter.

In the antigen detection method of the present invention, even if a pure immunogen is not obtained, if a monoclonal antibody is obtained, a reaction system with extremely high specificity can be created. It is possible to achieve a highly sensitive reaction equivalent to that of

(Examples of the Invention) Hereinafter, the present invention will be explained in more detail with reference to Examples, but these Examples are not intended to limit the scope of the present invention in any way.

Example carcinoembryonic antigen
ntigen.

Quantification of CEA) ■) Preparation of anti-CEA monoclonal antibody sensitized latex 0.
Add anti-CEA to 1 volume of 5% polystyrene latex 5DL59 (manufactured by Takeda Pharmaceutical Co., Ltd.: diameter 0.9 μm) PBS suspension.
Add 1 volume of 10 μg/ml of monoclonal antibody (manufactured by Israel Biological Research), sensitize by stirring slowly at room temperature for 60 minutes, centrifuge to collect the sensitized latex, wash twice with PBS, A sensitized latex was obtained by suspending it in PBS containing 0.1% bovine serum albumin.

21 Quantification of CE A Dispense 0.025 ml of the diluent (PBS with 0.1% bovine serum albumin) into each type of ■-type microtiter plate, and add 0.025 ml of the diluted sample (serum) to the 16th point. 025m
1 and diluted 2° using a diluter.

On the other hand, 4 ng/ml standard CEA was operated in the same manner without dilution. Next, 0.025 ml of the sensitizing latex of l) was added and stirred, and then anti-CEA immune serum (goat serum), 20
After adding 0.02541 of the 0-fold diluted solution and stirring, the tube was allowed to stand at room temperature for 6 hours, and the presence or absence of aggregation at the bottom of the tube was determined.

At the same time, enzyme immunoassay (EIA) was quantified using CEA enzyme immunoassay reagent (Kainos). The results are shown in the table.

The results of the antigen detection method of the present invention are in good agreement with the EIA method,
Both sensitivity and specificity were satisfactory.

[Effects of the Invention] The antigen detection method of the present invention enables qualitative or quantitative determination of an antigen specifically and with high sensitivity using extremely simple means, and is extremely useful for clinical testing.

Claims (1)

[Claims] contacting an antigen with particles sensitized with a monoclonal antibody against the antigen and a polyclonal antibody against the antigen;
An immunological antigen detection method characterized by detecting an antigen by the agglutination reaction that occurs.