RU1820325C - Method of antibodies assay to leptospira - Google Patents

Abstract

Usage: medical biochemistry, serological methods for the diagnosis of infectious diseases. SUMMARY OF THE INVENTION: a complex of staphylococcus Covan 1, with hypermune leptospirosis serum, conjugated with catalase, is used as antigen. 1 tab.

Description

The invention relates to medicine, in particular to the serological diagnosis of infectious diseases.

The purpose of the invention is to increase the sensitivity of the enzyme immunoassay in the determination of leptospirosis antibodies. .

This goal is achieved by the fact that hyperimmune leptospirosis serum is pre-adsorbed onto a suspension of inactivated staph cells of the strain Covan 1 and the whole complex is labeled with an enzyme catalase.

The claimed method differs from the prototype in a combination of new conditions: the use of staphylococcus cock to orient antibodies to leptospira and labeling of the complex, staphylococcus antibodies to leptospira with catalase enzyme.

These features make it possible to improve the conditions for the binding of antibodies to the antigen on the plate, which increases the sensitivity of the method, increases the catalase content on the complex, which also increases the sensitivity of the method, and the determination of antibodies to leptospira in serums, regardless of their species origin.

When studying the patent literature on the formulation of the enzyme-linked immunosorbent assay when determining antibodies to leptospira, similar features of the claimed method were not found by the authors.

Example. Preparation of conjugate by the proposed method. To 1 ml of 20 billion suspension of heat-inactivated inactivated in a boiling water bath for 5 min staphylococcus strain Covan 1 add 1 ml of leptospirosis serum at a 1:20 dilution. The mixture was stirred and kept for 60 minutes at 40 ° C, after which it was washed three times with 0.9% sodium chloride solution by centrifugation at 3000 rpm for THEN min. The antibody-loaded staphylococcus pellet is resuspended to 1 ml and catalase is added in a volume of 1 ml at the rate of 5 mg / ml, mixed and 0.1 ml of a 1% aqueous solution of glutaraldehyde is added. The mixture was kept at room temperature for 3 hours and washed three times with a 0.9% sodium chloride solution containing 0.05% tween-20. The precipitate was suspended in 5 ml of a 0.9% sodium chloride solution containing 0.05% tween-20.

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We used catalase to label staphylococcus-anti-leptospiram complex. US production Calbiochem activity 8512 IU / mg.

In parallel, horseradish peroxidase horseradish with natiglobulinriv serums (Gorky NIIEM) were prepared according to the known method, and in addition, a commercial test system (Stavropol NPO Allergen) was used to determine antibodies to leptospira.

The tablets of the Leningrad Polymer Plant were applied with a leptospirosis rhodospecific antigen at a dose of 10 μg / ml. The plates were held for at least 24 hours at room temperature and washed three times with 0.9% sodium chloride containing 0.05% Tween-20. In addition, tablets sensitized with a leptospirosis rhodospecific antigen, manufactured by NPO Allergen, were used.

Dilution workers of the resulting hogs were determined by direct ELISA (plate with adsorbed anti-tag + konogate in dilutions from 1:50 to 1: 3200). The result of ELISA was recorded visually.

When setting up the main experiments, homologous and heterologous sera from patients diagnosed with leptospirosis were layered on tablets with adsorbed antigen. shigellosis, escherichiosis. bacteriologically confirmed, and hyperimmune leptospirosis, shigellosis and Escherichia serum. Serum was layered in a volume of 0.1 ml in a diluted from 1:25 to 1: 51200. After 60 minutes of exposure, the plates were washed three times with 0.9% sodium chloride containing 0.05% Tween-20.

Then, konyogates were added to the tablets in working dilution: 1: 800 of their own preparation, and commercial ones, according to the instructions attached to the test system. After 60 minutes of exposure, the plates were washed three times with 0.9% sodium chloride containing 0.05% Tween-20.

To develop catalase, 0.025 ml of a 0.3% solution of permanganate were added to the tablets, and before that, in 30 minutes, a 0.0015% solution of hydrogen peroxide was added. When

In the presence of catalase, hydrogen peroxide decomposes and the potassium permangandt does not lose its violet color. In the presence of hydrogen peroxide, potassium permanganate changes color to bright yellow in a few seconds. The peroxidase was developed using a chromogenic mixture consisting of 5-aminosalicylic acid and hydrogen peroxide. In case of presence

peroxidase color in the wells - brown or brown-pink. If not, the color is slightly pink or colorless. The development of the enzyme in the test system was carried out according to the instructions.

. The comparative sensitivity of the determination of antibodies and the specificity of the proposed method are shown in the table.

The comparative sensitivity of the methods showed the advantages of the proposed

method 2-4 times compared with the known method for the determination of antibodies to leptospira. The results of the reaction after the application of potassium permanganate are clear, appear after a few seconds and last for several days.

The specificity of the proposed method was verified by adding homologous and heterologous to tablets with adsorbed leptospirosis antigen.

antisera (table). As shown, the specificity of all the studied methods turned out to be quite high and similar.

Thus, we have developed a new technical solution for the formulation of the enzyme-linked immunosorbent assay based on catalase, immune leptospirosis serum and suspension of staphylococcus strain Covan 1, with a higher sensitivity for the determination of antibodies to leptospira.

Claims (1)

The claims A method for determining antibodies to leptospira, including adsorption of antigen on a solid phase carrier, incubation with immune serum and imaging agent, characterized in that, in order to increase the sensitivity of the method, a complex of hyperimmune leptospirosis serum adsorbed on inactivated staphylococcus cells of the Covan strain and conjugated with catalase is used as antigen. The sensitivity and specificity of metopes on the example of several sera