JPH0465982B2 - - Google Patents

Description

[Detailed description of the invention]

[Purpose of the invention] (Field of industrial application) Testing methods using agglutination reactions are used to determine blood types, but recently animal red blood cells and other carrier particles are sensitized to various antigens or antibodies. As a result, it has become widely used for testing various viral infections and for measuring physiologically active substances such as antibodies and antigens. The present invention aims to improve the analytical method using this agglutination reaction, which is frequently used in the field of immunochemistry, biochemistry, etc., and especially in the field of clinical testing. (Prior art) Conventional agglutination reactions include direct agglutination reactions that utilize antigens contained in the particles themselves, which are used in blood type determination, and sensitized carrier particles with antigens. There is an indirect agglutination reaction in which sensitized particles are agglomerated, but in either method, the agglutination reaction is carried out in the wells of a microplate or in a small test tube. In the analytical method using this agglutination reaction, even the microplate method, which allows measurement in the smallest amount, requires a reaction solution volume of about 50 to 125 microns. As a result, in the case of extremely small amounts, hemolytic reactions have been used, for example, to determine blood type. (Problems to be Solved by the Invention) The hemolytic reaction method is complicated, requiring five steps, and the analysis target is limited, while the agglutination reaction method requires a considerable amount of sample and reagent, albeit a small amount. This method cannot be used when only a trace amount of sample is available. [Structure of the Invention] The present invention is characterized in that the aggregation reaction can be analyzed in extremely small amounts by introducing water droplets into a liquid such as liquid paraffin and causing the aggregation reaction within the water droplets. This was done based on the discovery that even if this particular liquid was shaken or centrifuged, the water droplets would not disperse and could be fully used as a site for aggregation reactions. (Means for Solving the Problems) The present invention provides an immunochemical analysis method using an agglutination reaction, which is transparent and insoluble in water.
An aqueous solution where particles and a specimen coexist, which are inert to the agglutination reaction, have a specific gravity of 1 to 0.7, and are nonvolatile, at least in response to the presence of the physiologically active substance to be analyzed. form,
The present invention relates to a method for analyzing a physiologically active substance, which is characterized by inspecting the agglomeration state of the particles in the field. The liquid that forms the aqueous solution field must at least be transparent, insoluble in water, inert to the aggregation reaction, have a specific gravity of about 1 to 0.7, and be nonvolatile. be.
That is, the liquid must be transparent in order to examine the state of agglomeration, and it must be insoluble in water to allow the formation of aqueous fields. Furthermore, it must be inert to the aggregation reaction to prevent non-specific reactions, and must have a specific gravity of 1 to 0.7 to form an aqueous field within the liquid.
Furthermore, this liquid must be nonvolatile because it must exist until the agglutination reaction is completed and the test is carried out. The specific gravity of this liquid is especially 0.85
~1 is preferable. Preferably, this liquid also has a low viscosity. Examples of such liquids include liquid paraffin and silicone oil that satisfies these conditions. Among these, liquid paraffin is particularly suitable for the method of the present invention. The container for containing the liquid is not particularly limited, but a Terasaki plate or a well of a microplate can be used. Although the amount of liquid added to each well does not have to be constant, for example, in the case of a Terasaki plate, about 5 to 10 microns may be added per well. The field aqueous solution formed inside such a liquid may be the same as in a normal flocculation reaction, and is determined depending on the type of flocculation reaction. The type of aggregation reaction to which the method of the present invention can be applied is not particularly limited,
In addition to the direct agglutination reaction used to determine blood type, detection of antibodies caused by various infectious diseases such as Treponema pallidum, streptolysin O, streptokinase, mycoplasma, toxoplasma, thyroglobulin, lupus erythematosus,
Detection of various autoimmune antibodies such as rheumatism factor, detection of virus-related antigens such as _HBS ,
Detection of peptide hormones such as HGG and HPL,
It can be widely used to detect cancer-related antigens such as AFP, ferritin, and β ₂ -microglobulin. The particles that undergo an agglutination reaction in response to the presence of the physiologically active substance to be analyzed may be sensitized particles that are normally used for agglutination reactions. For example, gelatin particles sensitized with antigens, antibodies, etc. 57−
153658), polystyrene latex,
Bentonite, kaolin, charcoal powder, animal red blood cells, bacteria, etc. can be used. The smaller the particle size is, the more preferable it is, and particularly preferably about 1 to 3 μm. When using particles with a larger diameter than this, it is preferable to lower the particle concentration than in the case of a normal agglutination reaction in order to prevent non-specific reactions. Gelatin particles are particularly preferred because there is almost no non-specific reaction, fine particles of about 1 to 3 μm can be easily obtained, and furthermore, they are easily colored and the state of aggregation can be easily determined. The aqueous solution field is formed into a water droplet using an aqueous solution having a diameter of 0.5 to 10 μm, particularly 2 to 3 μm. This water droplet is formed by, for example, aspirating a predetermined amount of a particle suspension, a sample, or any other necessary liquid into a microsyringe, and injecting each liquid one after another into the first droplet. All you have to do is let it happen. After injection, the aqueous solution may be mixed by shaking or the like, and after being left for a predetermined period of time, the state of aggregation may be inspected. The inspection may be observed with the naked eye using an optical microscope or the like, or may be automated using an autoanalyzer or the like. (Function) When the amount of aqueous solution is several μ, it evaporates instantly on paper, but this evaporation is prevented by surrounding it with liquid such as liquid paraffin.
Moreover, this liquid also fulfills the function of allowing the droplets of the aqueous solution to exist stably without being dispersed. [Effects of the Invention] The agglutination reaction can be carried out in an extremely small amount of aqueous solution of several μm, and as a result, only extremely small amounts of specimen and reagent are required. Since the agglutination reaction method is much easier to operate than methods using hemolysis reactions, this ultra-trace analysis has become easy to perform. In addition, even in the indirect agglutination reaction method, it has become easier to judge based on the formation of aggregates as in the direct agglutination reaction method. [Example] Lymphocyte typing was performed on serum lymphocytes and brain extracts. Separate various serum samples using Ficoll-Conray specific gravity centrifugation and collect lymphocytes using 28kHz ultrasound.
Processed for minutes. Both the ultrasonicated lymphocytes and the separately prepared brain extract had a particle size of 1 to 3.2 μm.
The cells were sensitized to red-colored gelatin particles (manufactured by Fujirebio Co., Ltd.) or polystyrene latex particles (manufactured by Takeda Pharmaceutical Co., Ltd.) with a particle size of 0.5 μm. The sensitization method is 2ppm for gelatin particles.
In the case of polystyrene latex particles, they were suspended in a 0.2M glycine buffer pH 8.2 at a concentration of 0.25%, and sensitization was carried out using a conventional method. Add about 5μ of liquid paraffin with a specific gravity of 0.875 to each well of the Terasaki plate, and add 1μ of antiserum to this.
And 1μ of the above 0.125% sensitized particle suspension was injected with a microsyringe so as to form one droplet. After stirring this for 5 minutes with a micro mixer, it was left at room temperature for 30 minutes, and the aggregation state of each well was adjusted to 40 to 40 minutes.
They examined it using a 100x optical microscope and determined the type. The results obtained are shown in the table below.

[Table] The measurement results obtained using the conventional hemolytic reaction method were completely consistent with those obtained using gelatin particles.

Claims (1)

[Claims] 1. In an immunochemical analysis method using an agglutination reaction, at least the analytical substance is transparent, insoluble in water, insoluble in the agglutination reaction, has a specific gravity of 1 to 0.7, and is nonvolatile. A method for analyzing a physiologically active substance, which comprises forming an aqueous field where particles and a specimen coexist in an aggregation reaction depending on the presence of the target physiologically active substance, and inspecting the aggregation state of the particles in the field.