UA48561A - Diagnostic immunoenzyme assay kit providing for detecting antibodies against lepstospira in human and animal blood serum - Google Patents

Abstract

The invention relates to immunochemistry, medicine, veterinary medicine and may be used for immunological screenings of the human and animal blood serum with the aim of detecting the antibodies against the lepstospira. The diagnostic immunoenzyme assay kit is based on the antigens from the purified cultures of leptospira strains (493 Poland, Kabura, Perepelicyn, Pomona, Moskva V, Hond Utreht IV, M 20, Bratislava and Wijnberg). The antigens are absorbed onto the surface of the polystyrene plates. The conjugates used comprise the enzyme-labeled anti-species antibodies (anti-human IgM or anti-cattle, anti-pig, anti-horse etc.) providing for detecting the antibodies against the lepstospira in human and animal blood serum. The enzyme reaction is developed with the substrate-chromogen complex.

Description

Description of the invention

The invention belongs to immunochemistry, medicine, veterinary medicine and can be used for conducting immunological studies, diagnostic analyzes of human or animal infection with leptospirosis.

A well-known test system for detecting antibodies (AT) against leptospirosis in blood sera is based on the microagglutination reaction (PMA) (1). When carrying out PMA, live cultures of leptospira of different serological groups at the age of 5 - 15 days without signs of agglutination and lysis are used for antigens; when examining the culture liquid under a 70 microscope, 70 - 100 microbial cells should be counted in one field of view at magnifications of 20 x 10 or 40 x 10. The serum is examined in four dilutions - 1:50, 1:100, 1:500 and 1:2500 . The reaction is placed in agglutination plates. 100 μl of each serum dilution is poured into a separate row consisting of 3, 7 - 15 wells, depending on the number of leptospira cultures (which act as antigens) used in the reaction. Each culture-antigen is introduced in 10 µl in 4 wells with different dilutions of serum. After adding 72 antigens, the plates are shaken and kept in a thermostat at a temperature of 30°C for an hour. The control is a mixture of the leptospire culture with a physiological solution (0.8595 sodium chloride solution). Leptospores in the control must be mobile, without signs of lysis and agglutination. The reaction is taken into account by microscopy of drops from each well in the dark field of the microscope.This method is dangerous because live cultures of leptospores are the antigen, and the method is inefficient: only 20-30 sera can be analyzed under the microscope in one working day.

The invention is based on the task of creating an immunoenzymatic test system based on antigens isolated from purified cultures of leptospora strains (493 RoYapa, Kariga, Regereyisup, Rotopa, MozKma M, Nopa

Ben M, M 20, Vgaiwiama and UMI|prego); antigens are absorbed on the surface of polystyrene tablets. In the composition of conjugates, anti-species antibodies are used (anti (human YAM or anti-cattle, anti-horse) or protein A, 22 labeled with an enzyme. The enzymatic reaction is determined using a substrate solution with a chromogen. In one setup, 88 sera can be analyzed in 2 hours

Example 1 Determination of antibodies against leptospora antigens in human blood serum.

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). -- 3o In three wells add 20 μl of negative control (K' - negative for leptospirosis blood serum "(human), and in the other two - 20 μl of positive control (K" - positive leptospirosis human blood serum).

Cover the strips with an adhesive film or a lid and incubate at a temperature of 37"C for 2 hours.

After 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. "

Into the wells of the strips, add 100 μl of a solution of anti-species conjugate - anti-IdM-human, labeled with peroxidase, and incubate at 37"C in a thermostat for 45 minutes.

After the end of the incubation, the wells are washed six times and 10 μm of the developer solution (substrate solution with chromogen) are added there and incubated at 18 - 227C in the dark for 10 - 25 minutes. « 20 Stop the color reaction by adding 50 μl of stop reagent to all samples. -at

No more than 1 minute 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 antibodies in the serum. The amount of antibodies in the serum determined using our test system was compared with the data obtained using the microagglutination reaction. The results of the conducted research prove the 10095th sensitivity of the proposed test system. Example 2 Determination of antibodies against Leptospira antigens in cattle blood serum was carried out similarly to example 1, but as an anti-species conjugate, an anti-cattle conjugate labeled with peroxidase was used; positive leptospirosis blood serum of cattle served as the positive control (K), while leptospirosis-negative blood serum of cattle served as the negative control (K).

Example C Determination of antibodies against Leptospira antigens in pig blood serum was carried out similarly

Me, example 1, but the conjugate of protein A with peroxidase was used as an anti-species conjugate; the positive - M control (K") was the positive leptospirosis blood serum of a pig, the negative control (KU) was the blood serum of a pig negative for leptospirosis.

Example 4 Determination of antibodies against leptospira antigens in horse blood sera was carried out similarly to example 1, but as an anti-species conjugate, antibodies against horse immunoglobulins labeled with peroxidase were used; a positive leptospirosis blood serum of a horse was taken as a positive control (K"), and a negative leptospirosis blood serum of a horse was used as a negative control (KU).

Example 5 Determination of sensitivity and specificity of the proposed test system.

Using the named test systems, blood sera were examined (20 definitely positive and 50 definitely negative sera). All sera were tested in four replicates with each test system according to the described method. All 20 positive sera showed the presence of antibodies against Leptospira, which is 10095 sensitivity. 50 negative sera turned out to be truly negative (specificity - 10095) in the proposed test system. In the microagglutination reaction compared, out of 20 definitely positive sera, 4 were negative (titer "1:50). The sensitivity of PMA was 8095. 65 Thus, the proposed test system provides detection of antibodies against Leptospira antigens. in sera of humans, cattle, pigs and horses. Simple and reliable in operation, shows high sensitivity and specificity.

References 1. KeiUu R.MU. Ieriozrigov p. Sograspy 5, Vapshey )/,

VaiYskiom M, (eds): RyPadei!rpia, Zatsypaegv, 1991, p. 1295 - 1302. 2. A.T. Mikhailov, V.N. Simirsky "Methods of immunochemical analysis in developmental biology". M. "Science",

Claims (1)

The formula of the invention The diagnostic immunoenzymatic test system for the determination of anti-leptospirosis antibodies in human or animal blood sera, in which anti-species antibodies are used as conjugates (anti (human IAM or anti-cattle, protein A, anti-horse, anti-pig), labeled with an enzyme , which differs in that it contains an immunosorbent with /5 applied antigens of purified cultures of leptospora strains (493 RoiYapa, Kariga, Regereiisup, Rotopa, MozkKma M, Nopa Shegen IM, M 20, VgaiIziama and UMI|prega). Official Bulletin "Industrial Property" Book 1 "Inventions, useful models, topographies of integrated microcircuits", 2002, M 8, 15.08.2002. State Department of Intellectual Property of the Ministry of Education and Science of Ukraine. 1 b 50 - 60 b5