RU2509306C2 - Method for obtaining erythrocytic antibody ovis diagnosticum for indirect hemagglutination reaction (ihar) to indicate ovis antigen in biomaterial - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention proposes a method for obtaining erythrocytic antibody diagnosticum for indirect hemagglutination reaction (IHAR) to indicate ovis antibody in biomaterial. The method is implemented by preparation of stabilised erythrocytes with further sensibilisation by positive ovis serum. In order to manufacture erythrocytic antibody diagnosticum, positive ovis serum for sensibilisation of stabilised erythrocytes is diluted in the ratio of 1:200-1:400, inactivated at 60°C during 30 minutes, and formalinised erythrocytes are sensibilised in the following ratio: one volume of 10% suspension of erythrocytes, one volume of positive ovis serum in the working dilution of (1:200-1:400), which is inactivated at 60°C during 30 minutes. Then, one volume of fresh water solution (0.1-0.11%) of water soluble alizarine blue indicator is added. Mixture is shaken and exposed during 60 minutes at 45°C, thus being stirred from time to time.

EFFECT: method allows obtaining erythrocytic ovis antibody diagnosticum having high activity and specificity.

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Description

The invention relates to the field of veterinary medicine, in particular to the production of an erythrocyte ovate antibody testum for an indirect hemagglutination reaction for indicating ovate antigen in a biomaterial.

In the literature available to us, we did not find data on obtaining ovum erythrocyte antibody diagnosticum for RNGA. Therefore, as an analogue, we used the antibody neutralization reaction (RNA) and the bacteriological method to indicate ovate antigen in the biomaterial.

RNat staging

The solvent of the test material and all the ingredients for PHAt is normal, inactivated rabbit serum diluted 1: 100 in a 0.85% NaCl solution; pH 7.1. The reaction uses a 2.5% working suspension of dry oat erythrocyte antigen diagnosticum diluted with a 0.85% NaCl solution. Reactions are placed in transparent polystyrene plates with wells in four rows: the first is experimental (8-10 holes), the rest are control (6 holes). The test material is titrated in a volume of 0.25 ml.

Then, 0.25 ml of the test material are added to 1 and 2 wells of the 1-experimental and second-control rows and diluted with index 2. In the third (control) row, 0.25 ml of brucellosis antigen is added and also diluted with index 2. As an antigen, a suspension of killed B.ovis at a concentration of 5x108 microbial cells is used. 0.25 ml of positive oat serum in a dilution corresponding to 4 serum (hemagglutinating) units (for example, 1: 800) are added to all wells of the 1st (experimental) and 3rd (control) rows.

The determination of the minimum hemagglutinating unit of specific serum is carried out before setting PHAt. For one serum (hemagglutinating) unit, the limiting dilution of oat serum in RNGA is taken (for example, 1: 3200), which causes a clear agglutination of red blood cells by 4 crosses.

0.25 ml of 1% normal rabbit serum is added to all wells of the second (control) row. In the fourth (control) row, 0.5 ml oat positive serum is diluted with an index of 2, starting with the dilution that was used in the first (experimental) and third (control) rows and the consistency of the dilution with 4 serum units is checked (for example , 1: 800). In the fourth row, the reaction should be positive not less than in 2-4 initial dilutions of serum. The tablets are shaken and placed in a thermostat at 37 ° -38 ° C for 2 hours. Then, 0.05 ml (1 drop) of a 2.5% suspension of oat antigenic erythrocyte diagnosticum is added to all wells of four rows. The tablets are shaken and left at room temperature for 3-4 hours. In the presence of ovic antigen in the test material, red blood cells precipitate in the form of a “button” or a small ring - the neutralization reaction is positive. If the antigen is absent in the test material, then specific antibodies added to the reaction remain free and sensitized red blood cells stick together - the neutralization reaction is negative. This reaction is quite complex, 3-component (erythrocyte antigenic ovis diagnosticum + positive ovum serum + test material), however, it is simpler in comparison with pathological material.

The advantage of RNAt lies in the possibility of detecting antigen in pathological material unsuitable for bacteriological examination.

The disadvantages of RNAT are its weak sensitivity, the time required for its execution to indicate the B.ovis antigen in the biomaterial, and a rather complicated formulation technique. A bacteriological study for infectious sheep epididymitis is associated with a large expenditure of time required to identify the pathogen from the biomaterial and its further indication. Cultural features of B.ovis require special culture media and special conditions for growth. Therefore, we decided to develop a method for producing a highly active, specific, uncomplicated by manufacturing technology, simple by the technique of setting the reaction and low-time for conducting research oat antibody erythrocyte diagnosticum for RNGA, devoid of these disadvantages.

Increasing the activity and simplifying the technology for producing ovate antibody erythrocyte diagnosticum for RNGA is achieved by sensitizing stabilized red blood cells using conjugates of a water-soluble alizarin blue indicator (ASI) according to the parameters of the concentration of ingredients, pH, ionic strength of the medium, temperature and exposure.

The maximum sensitivity and specificity of RNGA is obtained with the following ratios of ingredients: one volume of 10% suspension of red blood cells, one volume of positive oat serum in working dilution (1: 200 1: 400) inactivated at 60 ° C for 30 minutes. Then add one volume of freshly prepared aqueous 0.1-0.11% ASI solution. The mixture is shaken and incubated for 60 minutes at 45 ° C, stirring occasionally. After this, the red blood cells are washed with a 0.85% NaCl solution containing 0.5% normal rabbit blood serum inactivated at 60 ° C for 30 minutes, pH 7.0-7.2. After washing, by centrifugation, a suspension of red blood cells is introduced into physiological saline in order to obtain a 2.5% concentration of red blood cells.

The resulting antibody ovum erythrocyte diagnosticum is highly sensitive in RNGA and specificity. Due to the effectiveness of the method with low antibody activity in working serum solutions - sensitin - it is possible to obtain antibody-based diagnostic diagnostics based on inactive serum.

Previously, the activity of the produced antibody diagnosticums was checked in the RNGA, in the study of oat antigen for RDSK (reaction of long-term complement fixation), prepared by LLC NPF Biocenter, Omsk, in limiting dilutions. In this case, the RNGA of the diagnosticum prepared by us revealed an oat antigen - 0.045 mg of antigen in 1 ml or 0.0225 mg in 0.5 ml, since the reaction is put in a volume of 0.5 ml.

The specificity of RNGA diagnosticum was studied by examining lymph nodes and organs from 10 healthy rams (240 objects) and 4 ewes vaccinated with a vaccine from B.melitensis Rev-1 strain (60 objects). A total of 300 samples were examined, and in all cases negative results were obtained, which confirms the high specificity of RNGA with our ovate antibody erythrocyte diagnosticum.

Having ascertained the specificity of RNGA with antibody erythrocytic ovis diagnosticum (the Caspian zonal NIVI), we examined pathological material (lymph nodes and parenchymal organs) of 10 rams (53 objects) from an economy poor for infectious epididymitis and 20 guinea pigs (123 objects) experimentally infected with culture B.ovis. The study was carried out by the bacteriological method, RNAT and RNGA with antibody diagnosticum of the Caspian zonal NIVI (table).

An antibody test ovum erythrocytic diagnosticum was tested in comparison with the neutralization reaction of antibodies and the bacteriological method. The results of the study are presented in the table.

The results of the study of RNGA, RNAT, bacteriological method of biological material from animals in various epizootic situations

Groups of animals The number of investigated Highlighted Crops Positive pH Positive RNGA with a diagnosis. Casp. ZNIVI alive's objects Total % Total % Total % one. Sheep-producers from farms that are unfavorable for infectious epididymitis 10 53 13 24.5 24 45,2 32 60.3 2. Guinea pigs infected with B.ovis culture twenty 123 9 7.5 62 50,4 120 97.5 3. Single brucellosis standard antigen for RA, RSK and RDSK (S-forms) - one - - neg. - neg. - four. Antigens made from Brucella strains 16/4 and 1096 (R-forms) - 2 - - 2 one hundred 2 one hundred 5. Biofactory B.ovis antigen (R-form) - one - - one one hundred one one hundred Total: thirty 180 22 12.5 89 48.9 155 86.4

As can be seen from the table, when examining 176 objects of pathological material from 30 animals from groups with different epizootic situations, positive results in RNGA were obtained in a much larger number of cases than the bacteriological method and RNAT, which indicates a higher sensitivity of RNGA with antibody diagnosticum.

The sensitivity and specificity of RNGA with ovate antibody erythrocyte diagnosticum was checked by examining a single brucellosis antigen for PA, PCK and RDSK (S-form), antigens made from B. abortus 16/4, 1096 (R-form) strains and oat antigen of biofactory preparation .

The test showed high activity in the study of homologous (oat) antigen, while negative results were obtained with a single brucellosis antigen for PA, PCK, RDSK (S-form).

In addition, it was found that ovate antibody diagnosticum in RNGA also reveals brucellosis antigen in the R-form, since B.ovis is in a persistent R-form.

Literature

Abutalipov A. Comparative diagnostic efficacy of RID, RNAt and the bacteriological method for brucellosis. Bulletin of agricultural science of Kazakhstan, 1982, No. 5, pp. 75-77.

Romakhov V.A. Development and improvement of tools, diagnostic methods and system of measures to combat infectious sheep epididymitis and animal brucellosis. Diss. Ph.D. in the form of a scientific report, 1992.

Sadykov S.Zh. Diagnostic effectiveness of RNGA and RNAT in brucellosis, "Veterinary Medicine", 1974, No. 6, p.107-108.

Khairov S.G., Yusupov O.Yu. Erythrocyte Antigen Test in RNAT and RTNGA. Materials of jubilee. scientific and practical. conf. GNU Prikasp. ZNIVI, Makhachkala - 2003.P.42-44.

Chernysheva M.I. et al. Dry erythrocyte diagnosticum for the reaction of passive hemagglutination and RNA with brucellosis, ZhMEI, 1970, No. 12, p.171.

Claims (1)

A method for producing an erythrocyte antibody ovic diagnosticum for an indirect hemagglutination reaction (RNGA) for indicating a B.ovis antigen in a biomaterial, comprising preparing formalized ram red blood cells with subsequent sensitization with positive ovum serum, characterized in that for the first time positive ovine serum for loading formalinized erythritol is diluted with 1: -1: 400, inactivate at 60 ° C for 30 minutes, add one volume of freshly prepared 0.1-0.11% solution of water-soluble alizarin of another indicator (ASI), the mixture is shaken and incubated for 60 min at 45 ° C, stirring periodically, after which the red blood cells are washed with a 0.85% NaCl solution containing 0.5% normal rabbit blood serum inactivated at 60 ° C within 30 minutes, pH 7.0-7.2, after washing, by centrifugation, a suspension of red blood cells is introduced into physiological saline in order to obtain a 2.5% concentration of red blood cells.