Method for production of immune serum to virulent strain bacillus anthracis antigen

FIELD: medicine microbiology.

SUBSTANCE: claimed method includes administering before contamination to the animal subsequently in right pope then after 14 days in left pope 0.2 ml of non-complete Freund's adjuvant with equal volume of physiological salt solution. Subsequent administering after 14 days in right pope up to 10 LD₅₀ of Bacillus anthracis 81/1 spore dredge doesn't cause animal death for long period (monitoring time is 35 days).

EFFECT: immune serum for screening of anthrax diagnosis preparation.

1 dwg, 1 tbl, 8 ex

The invention relates to medical Microbiology and concerns obtaining immune sera from animals infected with a virulent strain of .anthracis.

You know that Guinea pigs are highly sensitive to the causative agent of anthrax and die within a short period of time (3-4 days) after infection. Therefore, to study the dynamics of antibodies or specific antigenemia usually use avirulence (odnopetlevye) strains of this organism (Detection of specific antigens in experimental anthrax. Abalkin V.A., Sergeev L.V., Cherkasy B.L. // Ukr. Microb., Biol., Epidemiol. 1989, No. 12.-P.63-68).

You can use cows, which infect large doses of the pathogen (specific and nonspecific Factors of protection from cows experimentally infected with anthrax. Buravceva N.P., Rakitin EL, Alapin NM, etc. //current. questions immunodiagnosis. especially dangerous infections. Part 1., Stavropol, 1986).

The disadvantage of the first method is that odnopetlevye strains cause formation of antibodies to specific antigens, which does not allow to identify the full spectrum of antibodies in the sera of animals. This is possible only when immunization virulent strain of this organism. The second method is expensive.

The closest analogue of the proposed method is to publish "Factor is specific and nonspecific protection of cows, experimentally contaminated with anthrax".

The purpose of the invention is obtaining immune sera from Guinea pigs after injection animals spore suspension of a virulent strain .nthracis.

This goal is achieved by the fact that Guinea pigs before infection alternately, first in right groin, then after 14 days in the left groin injected 0.2 ml incomplete adjuvant's adjuvant (NAF) with an equal volume of saline (experimental group).

The control group of animals injected with 0.4 ml of physiological solution.

14 days after last injection NAF animals of both groups infect the introduction of the right groin 20, 200, 2000 dispute .nthracis 81/1 (5 animals per dose).

Animals of the control group infected with 200 and 2000 dispute, die within 3-4 days, while the animals of the experimental group infected with these doses of spores remain alive for 35 days (observation period). Bacteriological examination of organs and blood for 21 and 35 days, the anthrax was not selected.

In the reaction, immunodiffusion in gel (REED) with the cultural filters (KF) .nthracis 81/1 (tox⁺cap⁺) STI (tox⁺cap^-) 81/1/1TR (tox^-cap^-) titers of the sera of the animals of the experimental group was 1:4-1:16.

In rnga with antigen-based assays, prepared using KF .nthracis TIS, Davis (tox^-cap⁺), 81/1TR Titus is s antibodies sera of these animals ranged from 1:80-1:640, with desencibiliziruuchee erythrocytes - 1:20.

The specificity of RNA confirmed RTHA and Rat.

The proposed method for anthrax serum from Guinea pigs infected with virulent strain may be useful for estimating diagnostic products.

Examples of specific performance, confirming the implementation of the proposed method

Example 1. Introduction animals incomplete adjuvant-blockers

An equal volume of incomplete adjuvant's adjuvant and 0.85 per cent sodium chloride solution was mixed using a medical syringe and injection needle to obtain a stable emulsion. 0.4 ml of the emulsion was injected Guinea pigs in the right groin. After 14 days of such emulsion, these animals were injected in the left groin.

Control group animals were injected from 0.4 ml of 0.85% sodium chloride solution.

Example 2. Getting spore suspension .nthracis 81/1

.nthracis 81/1 were sown on agar of Hottinger, cups were incubated at 37°C for three days, then at room temperature for three days. The grown culture was removed by a loop in a test tube, which was made of 0.85% sodium chloride solution. Biomass suspended with a pipette and warmed up in the 80°10 min, re-suspended after warming up, left in the refrigerator (4°C) for 12 hours.

The remaining suspension was aspirated and was titrated to 0.5 is l 4.5 ml of saline, sown 0.1 ml of agar Hottinger to determine the concentration of viable spores.

Example 3. Infection of Guinea pigs disputes .nthracis 81/1

On the day of infected animals after 14 days after the last injection NAF spore suspension was simulated again to determine the concentration of spores. Animal experimental and control groups were injected in the right groin, 20, 200, 2000 spores in a volume of 0.5 ml.

Example 4. Bacteriological examination of dead animals

Usually within 3-4 days died Guinea pigs of the control group infected with 200, 2000 spores .nthracis 81/1, and part of the animals infected with 20 spores of this organism.

In the experimental group during this time has lost part of Guinea pigs infected with 2000 dispute, and animals infected with 20 and 200 spores .nthracis 81/1 remained alive for a long time (observation period of 35 days).

LD₅₀.nthracis 81/1 for the animals of the control group was about 20 spores, animals of the experimental group - 2000 dispute (see table).

Upon examination of dead animals was noted swelling at the injection of the pathogen, at the showdown - blood circulation with hemorrhage in the liver and spleen.

When sown on agar of Hottinger prints the internal organs of these animals were allocated .nthracis, which was identified to sensitivity to phage To test and pearl necklaces.

Example 5. Bacteriol the ecological survey of surviving after infection of Guinea pigs, the blood of animals

At 21 and 35 days surviving Guinea pigs after chloroformiate been opened. Blood was taken from the heart. Shelter and prints internal organs were seeded on agar of Hottinger.

At the opening 21 and 35 days after infection of the internal organs of animals were unremarkable. .nthracis of internal organs and blood of these animals was not provided.

After verifying the sterility of the blood serum of these animals was aspirated iactiveaware at 56°With 30 minutes To inaktivirovannye sera was added 1% thimerosal 1:100 and stored in the refrigerator before use in the reaction of indirect haemagglutination (RGA) and the reaction immunodiffusion in gel (REED).

Example 6. Obtaining antigenic erythrocytic diagnosticums

For sensitization formalizovannyi Anisimovna sheep erythrocytes were used for optimal sensitization cultivation culture filtrates (CF) of different genotypes .nthracis.

Culture filtrates .nthracis STI, 81/1TR, Davis received on previously described methodology (Barkov A. M., ALEXANDER Lipnitsky, Evteeva EV isolation and antigenic characterization of the components of culture filtrate of Bacillus nthracis. //Biotechnology, 2000. No. 6.-P.27-33).

Sensitization of erythrocytes was performed according to (Bushanan T.M., J.C. Feeley, Haues P.S., P.S. Brachman "Anthrax inderect hemagglutination test //J.Immunol, 1971.-Vol.107.-P.2631-2636").

Example 7. Check in rnga active the STI and specificity of sera of Guinea pigs, surviving after infection .nthracis 81/1.

Rnge set according to the standard technique in the volume of 0.025 ml as diluent used 1% normal rabbit serum in saline solution pH of 7.2. Test serum was titrated in the diluent with 1:10 to 1:1280. After making diagnostics in the volume of 0.025 ml dice were shaken, incubated at 37°With 40 minutes the Result was taken into account within 20 minutes

The specificity of RNA confirmed controls: diluent + erythrocyte diagnosticum; serum studied Guinea pigs + desensibilisation Anisimovna erythrocytes.

Titers of sera obtained at 21 and 35 days after infection with spores .nthracis 81/1, accounted for diagnosticum based on the KF. .nthracis STI 1:320-1:640; for diagnostics based on KF .nthracis 81/1TR and Davis - 1:80-1:320 (see table).

The specificity of RNA also confirmed RTHA and Rat.

Example 8. Activity of sera of Guinea pigs surviving after infection .nthracis 81/1 in REED

Serum of Guinea pigs were active in different REED with KF different genotypes .nthracis and formed a zone of precipitates in the division 1:4-1:16. (see drawing).

Thus, the preliminary introduction of cavies NAF leads to prolonged survival of animals after infection 10 LD₅₀.nthracis 81/1 with the formation of antibodies to various antigens is m virulent strain .nthracis.

The use of the sera of these animals will be useful for (selection) assessment of anthrax diagnostic products.

The method of obtaining immune serum to antigens of virulent strains of Bacillus anthracis, characterized in that the Guinea pigs before infection alternately, first in right, then after 14 days in the left groin injected 0.2 ml incomplete adjuvant's adjuvant with an equal volume of physiological solution, within 14 days after the last injection incomplete adjuvant's adjuvant, in the right groin Guinea pigs injected up to 10 LD₅₀spore suspensions of Bacillus anthracis 81/1, all infected animals survive up to 35 days (observation period).