RU2491553C2 - Method of production of r-brucellar erythrocytic antigen for indirect hemagglutination test (iht) - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: method of production of R-brucellar erythrocytic antigen for indirect hemagglutination test (IHA) includes production of formalinised red blood cells of sheep, their sensibilisation with sensitine obtained by growing bacterial mass of brucella, its washing-off with the hypertonic solution of sodium chloride, inactivation, extraction and separation of sensitine by centrifugation. At that the antigen is taken out from the strain B.abortus 16/4 by acting on the bacterial cells with 0.5% concentration of sodium dodecyl sulfate at the temperature of 68-70°C for 60 minutes.

EFFECT: invention enables to produce a diagnostic preparation of high performance for diagnostics of brucellosis in animals.

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Description

The invention relates to the field of microbiology, and in particular to methods for the manufacture of diagnostic preparations used in serological studies in veterinary medicine and can be used in medicine.

A known method of preparing an antigen for differential serological diagnosis of brucellosis in animals, including obtaining the bacterial mass of strain B. abortus R-1096 and its inactivation by gamma radiation [SU 946039 A, 05.02.79,]. However, the antigen obtained in this way is used only for the formulation of the agglutination reaction and the complement binding reaction.

There is also known a method of producing an erythrocyte antigen for an indirect hemagglutination reaction in brucellosis, including the preparation of formalized ram erythrocytes, their treatment with sodium dodecyl sulfate at a concentration of 1% at 50-60 ° C for 30 minutes, followed by sensitization with sensitin obtained by growing the bacterial mass of brucella .abortus 19, washing it with a hypertonic solution of sodium chloride, inactivation, extraction and separation of sensitin by centrifugation [RU 2283498 C1, 02.22.2005].

This method of obtaining brucellosis antigen for RNHA with brucellosis does not allow to obtain an antigen for the detection of R-hemagglutinins in blood serum, because for its manufacture use the brucella strain in S-form.

The technical result of the invention is to obtain an active and specific R-brucellosis erythrocyte antigen for detection of serum R-brucellosis antibodies in an indirect hemagglutination reaction (RNA).

The technical result of the method of manufacturing R-brucellosis erythrocyte antigen for the indirect hemagglutination reaction is achieved by preparing formalized ram erythrocytes, followed by sensitization with sensitin obtained by growing the bacterial mass of B. abortus 16/4, washing it off with a hypertonic solution of sodium chloride, inactivation, sensing extraction , exposure to bacterial cells with a 0.5% concentration of sodium dodecyl sulfate at a temperature of 68-70 ° C for 60 minutes.

The method of implementation. The three-day agar culture of Brucella vaccine strain B. abortus 16/4 is washed off with a 12% sodium chloride solution, filtered and autoclaved at 1 atm. (120 ° C) for 20-30 minutes, bring the concentration of brucella to 40-50 billion m. in 1 ml of 12% NaCl solution. To the bacterial suspension add 0.5% sodium dodecyl sulfate, after which it is heated in a water bath at a temperature of 68-70 ° C for 60 minutes, with periodic stirring. The suspension is decanted by centrifugation at 8-10 thousand rpm for 60 minutes, the extract obtained is used to sensitize formalized ram red blood cells.

Sheep erythrocytes are formalized according to the method of R. Weinbach (1958), while the formaldehyde concentration during the processing of red blood cells is 1.5%, and the ratio of fixative to red blood cells is 0.375: 1. The obtained suspension of red blood cells with formaldehyde is transferred to a thermostat and formalized at a temperature of 37-38 ° C for 18-20 hours. After formalinization is completed, erythrocytes are washed from excess formalin 3 times with a 10-fold volume of physiological saline in a centrifuge at 1000-1500 rpm for 10-15 minutes (Karalnik B.N. Erythrocyte diagnostics. - M., Medicine. - 1976. - 164 p.).

Table 1 presents the results of exposure to the bacterial mass in order to extract sensitin of different concentrations of sodium dodecyl sulfate.

Table 1 Effect of sodium dodecyl sulfate concentration on the activity and specificity of R-brucellosis erythrocyte antigen Sodium dodecyl sulfate concentration The amount of antigen per 1 ml of 5.0% suspension of red blood cells RIGA titer with R-brucellosis serum Negative cattle serum with 1:50 Saline Control 0.1% 0.25 - - - 0.5 - - - one - - - 2 - - - 3 - - - 0.25% 0.25 - - - 0.5 - - - one 1: 100 ++++ - - 2 1: 400 ++++ - - 3 1: 800 ++++ - - 0.5% 0.25 1: 1600 ++++ - - 0.5 1: 3200 ++++ - - one 1: 6400 ++++ - - 2 1: 12800 ++++ 1: 100 ++++ - 3 1: 25600 ++++ 1: 200 ++++ ++++ 1,0% 0.25 1: 25600 ++++ 1: 400 ++++ ++++ 0.5 1: 25600 ++++ 1: 400 ++++ ++++ one 1: 25600 ++++ 1: 400 ++++ ++++ 2 1: 25600 ++++ 1: 400 ++++ ++++ 3 1: 25600 ++++ 1: 400 ++++ ++++

An analysis of the data in Table 1 shows that the optimal concentration of detergent in the manufacture of R-antigen is 0.5% sodium dodecyl sulfate, while a specific erythrocyte antigen with a given activity of not less than 1: 3200 in titration with R-brucellosis serum can be obtained at a ratio of formalized red blood cells and sensitin 1: 0.5. When using a 0.25% concentration of dodecyl sulfate in the process of extracting sensitin, the diagnosticum does not have sufficient activity. When using sodium dodecyl sulfate at a concentration of 1.0%, the erythrocyte diagnosticum becomes non-specific, and positive results were obtained with negative cattle serum at a dilution of 1:50 and above and with physiological saline.

The sensitization of formalin erythrocytes is carried out by the optimal dose of sensitin, which is determined by titration using R-brucellosis agglutinating serum. When titration of sensitin to sensitize red blood cells to 1 ml of 5.0% suspension of red blood cells add it in quantities of 0.25; 0.5; 1.0; 1.5; 2; 3.0 ml The results of the specificity test of R-brucellosis erythrocyte antigen are presented in table 2.

table 2 The result of the specificity test of R-brucellosis erythrocyte antigen Examined blood sera The number of test samples Serum dilution Research result cattle unvaccinated against brucellosis from brucellosis-friendly farms 505 1:50 negatively healthy rabbits twenty 1:10 negatively healthy guinea pigs 26 1:10 negatively rabbits hyperimmunized with the vaccine strain B. abortus 104 M (S-form) 10 1:10 negatively cattle with brucellosis sick from brucellosis dysfunctional farms 79 1:50 negatively

From the data presented in table 2, it follows that the proposed method allows to obtain a specific R-brucellosis erythrocyte antigen, because allows to obtain negative results of RIGA in healthy and sensitized brucellosis S-antigen of animals. The test results of the activity of R-brucellosis antigen are shown in table 3.

Table 3 The test results of the activity of R-brucellosis erythrocyte antigen No. p / p Object of study Research results Rnga RA with a single antigen (titer ME) RSK with a single antigen (dilution of blood serum) one blood serum of rabbits infected with B. abortus 54-70 (R-form) 1: 2560 - - 2 1: 2560 - - 3 1: 2560 - - four 1: 3200 - - 5 1: 200 - - 6 1: 1600 - - 7 1: 1600 - - 8 1: 3200 - - 9 1: 800 - - 10 1: 100 - - one serum of cattle immunized with a vaccine from strain B. abortus 82 (R S form) 1: 200 - - 2 1: 400 - - 3 1: 1600 50ME +++ - four 1: 800 - - 5 1: 400 - - 6 1: 3200 - 1: 10 ++ 7 1: 800 - - 8 1: 400 - - 9 1: 200 - - 10 1: 3200 100ME ++ -

From table 3 it follows that the R-brucellosis erythrocyte antigen has activity, so hemagglutinins in the blood serum of rabbits infected with the R-form of brucella were contained in the titer from 1: 100 to 1: 3200, in the blood serum of cattle immunized with a vaccine from the strain B. abortus 82 from 1: 200 to 1: 3200.

The sensitivity of R-brucellosis erythrocyte antigen was determined by examining the blood serum of cattle grafted with a vaccine from strain B. abortus 82 in farms that were safe and dysfunctional for brucellosis (Tables 4, 5).

Table 4 The results of serological studies of blood serum of cattle inoculated with a vaccine from strain B. abortus 82, in brucellosis-free farms Study period (days) Farm number Samples Investigated Reacted in RNA with R-antigen RA + RSK with a single antigen abs. % abs. % thirty one 49 34 69,4 3 6.1 2 198 171 86.4 43 21.7 60 one 196 106 54.1 6 3,1 90 one 189 130 68.8 eleven 5.8 120 one 199 60 30.1 3 1,5 180 one 198 thirty 15,2 6 3 210 one 191 22 11.5 6 3,1 TOTAL: 1220 553 45.3 78 6.4

An examination of bovine brucellosis-free cattle immunized with a vaccine from strain B. abortus 82 showed that using R-brucellosis erythrocyte antigen over the course of 210 days, 11.5 to 86.4% of the responding animals were detected.

Table 5 The results of serological studies of blood serum of cattle inoculated with a vaccine from strain 82 in herds unfavorable for brucellosis Study period after vaccination (day) Number of goals Number of animals reacting in: RA + RSK + RNGA + RID with S-antigens in high titers RNGA with R antigen abs. % abs. % 60 539 31 5.8 25 80.6 90 504 16 3.2 fifteen 93.7 120 471 7 1,5 7 100.0 Total: 1514 54 3.6 47 87.0

The data in table 5 show that R-brucellosis antibodies in the blood serum of patients with brucellosis in animals vaccinated with a vaccine from strain B. abortus 82 (SR-form) can be diagnosed using R-brucellosis erythrocyte antigen made by this method, at different times after immunization animals.

The proposed R-brucellosis erythrocyte antigen has specificity and sensitivity, it is used in the laboratory of the GNU VNIIIBTZ when monitoring the immune response in cattle immunized with anti-brucellosis vaccines, as well as in the recovery of farms that are unsuccessful for brucellosis in order to preserve post-vaccination animals from unreasonable slaughter.

Claims (1)

A method of manufacturing an R-brucellosis erythrocyte antigen for an indirect hemagglutination reaction (RNGA), comprising preparing formalized ram erythrocytes, sensitizing them with sensitin obtained by growing the bacterial mass of brucella, washing it off with a hypertonic sodium chloride solution, inactivating, extracting and separating the sensor, the antigen is extracted from the R strain of B. abortus 16/4, affecting the bacterial cells with a 0.5% concentration of sodium dodecyl sulfate at a temperature of 68-70 ° C for 60 minutes.