JPH0456258B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an aqueous solvent for aggregation tests. A method for quantitatively measuring one or both of these by tracking antigen-antibody reactions or agglutination reactions based on immune reactions between agglutinating substances derived from certain types of animal, plant, or bacterial cells and biological proteins or red blood cells. is widely used in the field of clinical testing as hemagglutination reaction (PHA method), reverse passive hemagglutination reaction (RPHA method), bacterial agglutination reaction, latex agglutination reaction, etc. For example, to explain the PHA method, purified HBs antigen is used as a carrier for an agglutination test to sensitize sheep red blood cells to obtain HBs antigen-sensitized sheep red blood cells, and when this is mixed with a blood sample, an agglutination reaction occurs in an aqueous medium. It can be determined whether HBs antibodies are present in the sample depending on whether or not HBs antibodies are observed. In addition, if an agglutination reaction is observed, the sample is further diluted stepwise to determine the dilution ratio until the agglutination reaction can no longer be observed.
HBs antibodies can be measured quantitatively. In addition, in the RPHA method, purified HBs antibodies are used as a carrier for an agglutination test to sensitize mammalian red blood cells.
Detecting whether or not the sample contains HBs antigen by producing HBs antibody-sensitized red blood cells and checking whether an agglutination reaction is observed in an aqueous solvent when mixed with the sample. I can do it. Furthermore, certain viruses and avian red blood cells agglutinate red blood cells in an aqueous medium due to the action of the agglutinin of the virus. The concentration of the virus can be quantitatively measured by the presence or absence and strength of the agglutination reaction in this aqueous solvent. It is well known that many of these analytical methods that utilize agglutination reactions in aqueous media are simple and have high measurement sensitivity, and are therefore widely used for testing various antigens and antibodies. However, depending on the type of antigen or antibody to be sensitized, a clear agglutination reaction may not be observed in these aqueous solvents even though the agglutination carrier has been sufficiently sensitized. In quantitative tests, the agglutination reaction becomes weaker at low dilution ratios (at high concentrations of the corresponding antigen or antibody), and it may be misjudged that no agglutination reaction is observed (prozone phenomenon). To clarify aggregation reactions in aqueous solvents,
A known method for solving the prozone phenomenon is to lower the ionic strength of the aqueous solvent used for measurement. However, this is still not sufficient, and the emergence of an even better method has been awaited. An object of the present invention is to provide an aqueous solvent for aggregation tests that enhances weak agglutination reactions, allows for more clearly distinguishing agglutination reactions, solves the prozone phenomenon, and has extremely excellent detection sensitivity. The inventors have continued research to improve the measurement method for many years in order to improve the weak agglutination reaction and prozone phenomenon in this agglutination reaction. It was discovered that the zone phenomenon was significantly improved and the measurement sensitivity was improved, and the present invention was completed. That is, the present invention provides an aqueous solvent for an agglutination test based on an immune reaction, which contains dextran having an average molecular weight of 30,000 to 100,000 at a concentration of about 1% to 3% by weight. By using this aqueous solvent in preparing reagents for various aggregation reaction tests, the above-mentioned improvement in measurement sensitivity can be achieved. The average molecular weight of the dextran used in the present invention is 30,000 to 100,000. The amount added is 1 to 3
It is about % by weight. The lower limit of the amount added is the limit value of the detection sensitivity and prozone phenomenon suppressing effect, and the upper limit is the limit value of the decrease in detection sensitivity due to an increase in the negative value. As the aqueous solvent, any conventionally known aqueous solvent for agglutination tests can be used, such as water, physiological saline, various buffers (phosphate buffer, Tris-HCl buffer, borate buffer, glycine buffer), albumin Examples include solutions consisting of solutions, normal human and animal serum solutions, solutions of synthetic polymeric substances, surfactant-containing aqueous solutions, and combinations thereof. The pH of the aqueous solvent of the present invention is preferably about 6.5 to 9.0, and is preferably adjusted using a buffer. The concentration of the salt contained in the aqueous solvent of the present invention is preferably a relatively low concentration, and more preferably a solution isotonic with body fluids. As carriers for aggregation tests used in the aqueous solvent of the present invention, those known per se may be used, such as latex resins, rubbers, inorganic adsorbents, and particularly preferred carriers include animals (such as sheep, guinea pigs, Red blood cells from humans (types of humans, chickens, etc.) fixed with formalin, glutaraldehyde, etc. may also be used. It is advantageous to use such a carrier in the form of particles of 20 microns or less, particularly about 5 to 15 microns. Treatment for sensitizing such particulate carriers with antibodies or antigens can be carried out according to methods known per se. Experiments were conducted to prove the effectiveness in a flocculation test using the aqueous solvent of the present invention thus provided. HBe antigen-positive human serum is treated and purified using ammonium sulfate fractionation and affinity chromatography.
HBe antigen was obtained, and sheep red blood cells fixed with glutaraldehyde were sensitized with the purified HBe antigen to obtain HBe antigen-sensitized sheep red blood cells. On the other hand, as the aqueous solvent of the present invention, 0.1 M
A known solvent for the PHA method was prepared by adding normal animal serum and sheep red blood cell membrane components to a phosphate buffer (PH7.5). This solvent was prepared by adding dextran (average molecular weight 50,000 to 70,000) in the amount shown in Table 1, and as a control, a sample without any additive was prepared. Three citrated human plasma samples (1, 2, and 3 in the table) that were positive for HBe antibodies as measured by the Ouchterlony method (two-way immunodiffusion method) were
According to the PHA method, each of these three solvents was diluted 2-fold on a microplate, and the HBe antigen-sensitized sheep red blood cells obtained above were mixed into this dilution series. Separately, a specimen (4) negative for HBe antigen was similarly treated and tested. The volume of the mixed solution was 25μ of sample dilution solution and 25μ of HBe antibody-sensitized sheep red blood cells.The strength of agglutination (degree of agglutination) after 2 hours at room temperature was evaluated as 4, 3, 2, 1, and non-aggregation was evaluated. When expressed as 0, the results shown in Table 1 were obtained.

[Table] As is clear from Table 1, dextran
By adding % by weight, weak aggregation with an aggregation degree of 1 to 2 became strong agglutination with an agglutination degree of 3 to 4, and the prozone phenomenon observed in plasma samples No. 1 and 3 was almost completely improved. There is. Furthermore, sensitivity has also increased significantly. In addition, in the case of negative samples, the ratio was 1:4 or less, which is the same as when no additive was added, in any case, indicating that there was no effect on the negative samples. Thus, the aqueous solvent for aggregation tests of the present invention significantly enhances the aggregation reaction and increases sensitivity, making it possible to provide an excellent aggregation test method. The present invention will be specifically illustrated below using Examples. Example 1 Dextran (average molecular weight 50000-70000) 2.0g
And to 10 ml of 20 w/v% bovine albumin solution, add 0.1 M phosphate buffer with pH 7.6 to make 200 ml. An experiment similar to the above was conducted using this solvent, but there was no prozone phenomenon and the detection sensitivity was good.

Claims (1)

[Claims] 1. An aqueous solvent for an agglutination test based on an immune reaction, which contains dextran having an average molecular weight of about 30,000 to 100,000 at a concentration of about 1% to 3% by weight.