UA68240A - Immunoenzyme kit for assaying antibodies against mycobacterium bovis in blood serum of the cattle - Google Patents

Abstract

The immunoenzyme kit for assaying antibodies against Mycobacterium bovis in blood serum of the cattle consists of the immunosorbent containing the antigens of the purified Mycobacterium bovis cultures and the conjugate containing enzyme-labeled antispecies monoclonals (anti IgM and IgG of the cattle).

Description

Description of the invention

The invention belongs to immunochemistry, veterinary medicine and can be used for conducting immunological studies, diagnostic analyzes of cattle infection for tuberculosis infection.

A well-known test in Ukraine for detecting tuberculosis infection is based on the tuberculin test. In Russia, a test system was created for the detection of total antibodies to Musobrasiegisht (shregstiov in human blood serum

PI, which uses antigens of mycobacteria and non-oxidase conjugate of monoclonal antibodies to 70 human immunoglobulins C, A and M. Samples from patients before the analysis are pre-diluted 1:100 in a buffer for dissolving sera and only then used for analysis. This indicates insufficient purification of antigens from mycobacteria. Immunoenzymatic test system for determining antituberculosis antibodies in cattle is not produced in Ukraine.

The invention is based on the task of creating an immunoenzymatic test system based on 72 antigens isolated from purified cultures of Musopasiegisht Nomiz strains. Antigens are adsorbed on the surface of polystyrene tablets. In the composition of conjugates, monoclonal anti-species antibodies (anti-DM and anti-CAV), labeled with an enzyme, are used. The enzymatic reaction is determined using a substrate solution with a chromogen. 88 sera can be analyzed in one setup (2 hours).

Example 1

Determination of antibodies against antigens of mycobacteria that cause tuberculosis in blood serum of cattle.

In each well of the strips of the tablet, add 8 μl of serum dilution solution and 20 μl of samples of the tested sera, leaving 5 wells of the first row free (control wells).

Add 20 μl of negative control (K' - tuberculosis-negative human blood serum) into three wells, and 20 μl of positive control (K" - positive tuberculosis human blood serum) into the other two wells.

Cover the strips with an adhesive film or a lid and incubate at a temperature of 37°C for two hours.

At the end of the incubation, remove the contents of the wells using a washer or an 8-channel pipette and wash the wells four times with a solution for washing tablets.

100 μl of a solution of monoclonal anti-species conjugate - anti-IDM and anti-IdDO-BKV, labeled with peroxidase, is introduced into the wells of the strips, and incubated at 37 C in a thermostat for 45 minutes.

After the end of the incubation, the wells are washed six times and 10 µl of developer solution "I" (substrate solution with chromogen) is added there and incubated at 18-227C in the dark for 10-25Xmin. high school

The color reaction is stopped by adding 50 μl of stop reagent to all samples.

No more than 1 minute later. after stopping the color reaction, determine the optical density (OD) in the wells in the two-wave mode using a spectrophotometer. The OG value of the sample is directly proportional to the amount of c antibodies in the serum.

Example

Determination of sensitivity and specificity of the proposed test system.

Blood sera were studied (100 definitely positive and 100 definitely negative sera). All « 70 Serums were studied in four repetitions according to the described method. Out of 100 positive sera, 97 showed the presence of antibodies against the causative agent of tuberculosis, which is 9795 sensitivity. Out of 100 negative sera, 95 turned out to be truly negative (specificity - 9595) in the proposed test system. In the compared test system from Russia, out of 20 definitely positive sera, 2 were negative. The sensitivity was 9096.

The specificity of the comparative test system was 9590.

Thus, the proposed test system provides detection of antibodies against antigens of the causative agent of tuberculosis in cattle sera. Simple and reliable in operation, shows high sensitivity and specificity.

Sources of information (about) 1. AT-Tub-Best-strip. Demonstration of total antibodies (Ids, I9M, IdDA)

HF (plasma) of human blood. Diagnostic test systems! Novosibirsk, 2000. 2. A.T. Mikhailov, V.N. Simirsky "Methods of immunochemical analysis in developmental biology". M. "Nauka", vol. 1981.

Claims (1)

The formula of the invention is a diagnostic immunoenzymatic test system for the determination of anti-tuberculosis antibodies in blood sera. of cattle (VKR), which contains in the conjugates anti-species antibodies, labeled with an enzyme, which differs in that it contains an immunosorbent with applied antigens of purified cultures of the causative agent of tuberculosis (Musobrasiegisht roms), and as a conjugate, monoclonal anti-species antibodies (anti ИЯ9М and to КРХ), labeled with an enzyme that ensures the detection of antituberculosis antibodies in the blood sera of 60 cattle. Official bulletin "Industrial Property". Book 1 "Inventions, useful models, topographies of integrated microcircuits", 2004, M 7, 15.07.2004. State Department of Intellectual Property of the Ministry of Education and Science of Ukraine.