RU2493871C1 - Method for preparing antibody erythrocyte diagnosticum for indirect hemagglutination test (iht) for purposes of brucella indication in pathologic material - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to veterinary science, namely to preparing an antibody erythrocyte diagnosticum for brucella indication in a pathologic material. Substance of the invention involves a method for preparing the antibody erythrocyte diagnosticum for indirect hemagglutination test (IHT) for purposes of brucella indication in the pathologic material by preparing formalinised erythrocytes to be sensitised by a positive brucellosis serum; for the purpose of preparing the antibody erythrocyte diagnosticum the positive brucellosis serum for sensitisation of the formalinised erythrocytes is dissolved in ratio 1:50-1:100, inactivated at 60°C for 30 minutes; the formalinised erythrocytes are sensitised at: a residue of 2 volumes of 40% sheep erythrocyte suspension, 1 volume of 0.43-0.44% fresh amidol, 1.0-1.5 volumes of the positive serum dissolved in ratio 1:50-1:100 and kept for 28-32 minutes at 55-56°C.

EFFECT: invention enables preparing the high-activity and high-specificity brusellosis antibody erythrocyte diagnosticum.

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Description

The invention relates to the field of veterinary medicine, namely to obtaining erythrocyte brucellosis antibody diagnosticum for the reaction of indirect hemagglutination, with the aim of indicating brucella in the material.

The well-known "Method for producing erythrocyte antibody-based diagnosticum for indicating brucella in the test material", the essence of which is as follows. For the sensitization of red blood cells, positive brucellosis blood sera diluted with physiological saline 1: 80-1: 100 or physiological buffered saline inactivated at 64 ° C for 20 minutes with an optimum pH of 6.4-7.0 are used.

The optimal parameters of erythrocyte sensitization conditions by positive blood serum are as follows: 1 volume of pre-titrated concentration of amidol solution is mixed with 2 volumes of a 20% suspension of fixed red blood cells and 1 volume of positive brucellosis blood serum at a dilution of 1: 100, when incubated in a water bath at a temperature of 55 ° C for 25-30 minutes, with periodic shaking every 5-10 minutes.

Sensitized red blood cells are washed with one of the stabilizers (normal horse or rabbit serum), diluted 1: 100-1: 500, in a centrifuge at 1500-2000 rpm for 5-10 minutes twice, after which they are suspended to 0.75% the concentration of isotonic sodium chloride solution and is used for RNGA for the purpose of indicating brucella: Sh.A. Baramova, author. Diss., Ph.D., 1988.

The disadvantage of this method is the weak activity of the diagnosticum made by this method in the reaction of indirect hemagglutination (RNGA) when indicating brucella in the test material. In addition, this diagnosticum in some cases is spontaneously agglutinated in RNGA with normal rabbit serum, which is used as a stabilizer for breeding studied materials where there are no brucellas. Therefore, we decided to develop a method for obtaining a highly active and specific brucellosis antibody erythrocyte diagnosticum for RNGA, devoid of these disadvantages.

The aim of the invention is to increase the activity and specificity of brucellosis antibody erythrocyte diagnosticum for RNGA. This goal is achieved in that the sensitization of sheep erythrocytes (Weinbach stabilized in our modification) using the amidol conjugate is carried out according to the parameters of the concentration of ingredients, pH, ionic strength of the medium, temperature and exposure.

The maximum sensitivity and specificity in RNGA are obtained with the following ratios of ingredients: sediment from 2 volumes of a 40% suspension of sheep erythrocytes, 1 volume of a 0.43-0.44% solution of freshly prepared amidol and 1.0-1.5 volumes of positive serum blood diluted 1: 50-1: 100 in a 0.85% NaCl solution, pH 7.0-7.2 in the absence of phosphate buffer, inactivated at 60 ° C for 30 minutes, the mixture is stirred and kept in water bath 28-32 minutes at 55-56 ° C, shaking periodically every 5-6 minutes.

Sensitized red blood cells are washed off with a 0.85% NaCl solution containing normal rabbit serum at a dilution of 1: 50-1: 100 or horse serum at a dilution of 1: 220-1: 250, inactivated at 60 ° C for 30 minutes, pH - 7.0-7.2. At the end of centrifugation, suspensions of red blood cells are added to physiological saline in order to obtain a 2.5% concentration of red blood cells.

Preliminarily, the activity and specificity of the produced antibody diagnosticums is checked in the RNGA, in the study of a single brucellosis antigen for RA, RSK and RDSK in maximum dilutions.

Antibody diagnosticum was tested in comparison with a similar drug proposed by Sh.A. Baramova.

The activity and specificity of antibody-based diagnosticum produced by our method (antibody-based diagnosticum of the Caspian zonal NIVI) was tested in comparison with a similar drug proposed by Sh.A. Baramova (prototype).

To test the diagnosticum in RNGA, a single brucellosis antigen was studied for RA, CSC, and RDSK (Table 1).

Table 1 The results of checking the activity of diagnosticums in RNGA Red blood cell formalinization method Amidol Concentration (%) 0.24 0.27 0.30 0.32 0.34 0.36 0.38 0.40 0.43 0.44 Brucella detected (thousand sq.m. in 1 ml) According to Weinbach in our modification with diagnosticum Prikasp. ZNIVI 366.2 183.1 91.5 45.7 22.8 11,4 5.7 2,8 1.43 1.43 with analog 366.2 183.1 91.5 45.7 22.8 - - - - -

As can be seen from the data given in table 1, the diagnosticum we developed is more sensitive by 15.9 times in comparison with the analogue.

Antibody diagnosticum test in comparison with a similar drug manufactured by the method of Sh.A. Baramova

To test antibody diagnostics in the RNGA, suspensions of lymph nodes and organs were studied in healthy cattle (20 animals) and 30 in sheep. The results of the specificity of these diagnostic kits are presented in table 2.

Table 2 The results of the specificity test of antibody diagnosticum in RNGA Material (suspension) Number of goals Organ and lymph nodes examined Casp. ZNIVI VRNGA Analog in RNGA floor. neg. floor. neg. From organs and lymph nodes of a healthy cr. horn. cattle twenty 200 200 one 199 From organs and lymph nodes of sheep thirty 150 150 2 148

As can be seen from table 2, when testing in RNGA with an antibody-based erythrocytic diagnosticum manufactured according to our method (antibody-based diagnosticum Prikasp. ZNIVI), a negative result was obtained with all organs and lymph nodes of suspensions of healthy animals, which shows its high specificity, whereas by analogy in 3 cases a positive result, i.e. the prototype in some cases gives nonspecific reactions with a suspension of healthy animals.

In addition, in some cases, the analogue is spontaneously agglutinated in RNGA with normal rabbit serum, which is used as a stabilizer for diluting the test material to detect the causative agent of brucellosis, especially when the pH of the medium is 6.4, as recommended by Sh.A. Baramova.

It turned out that lengthening the time of sensitization of red blood cells to 32 minutes led to an increase in the sensitivity of indication of the causative agent of brucellosis, and with an increase in temperature above 28-32 ° C, with the duration of the contact of red blood cells with positive serum more than 40-60 minutes, spontaneous hemagglutination developed (Table 3) .

Table 3 The results of indicating the optimal temperature and exposure sensitization of red blood cells of positive blood serum (diagnosis. Prikasp. ZNIVI) Breeding positive brucellosis serum (sensitin) Exposure Temperature (min.) 45 ° C 50 ° C 55-56 ° C 60 ° C 28 32 40 28 32 40 28 32 40 28 32 40 1:40 - - - - - - - - - - - - 1:50 - - - - - - Dv Dv Spontaneous hemagglutination 1: 100 - - - Ds Ds Dh Dv Dv - // - - // - - // - - // - 1: 150 - - - Dh Dh Spontaneous hemagglutination - // - - // - - // - - // - Note: DS - diagnosticum, which showed weak sensitivity in RNGA; DCH - sensitive diagnosticum, but with a low titer; Dv - diagnosticum highly sensitive; Spont hemaggl. - a diagnosticum that showed spontaneous hemagglutination in RNGA; - a diagnosticum that showed a negative result in the RNGA.

Many researchers (Chernysheva M.N. (1970), Sadykov S.Zh. (1974), Abutalipov A. (1982), Romakhov V.A. (1992) and others) used the neutralization reaction of antibodies to detect brucellosis antigen in the material (RNAT) and bacteriological method. Therefore, we decided to conduct a comparative test of the diagnostic effectiveness of antibody brucellosis diagnosticum for RNGA (Prikasp. ZNIVI), antibody neutralization reaction (RNAT) and bacteriological method.

To this end, pathological material from a cattle brucellosis patient (7 animals), sheep (11 animals), milk infected with the causative agent of this disease (5 samples) and tap water infected with brucella (vaccine strain B.abortus 19) were examined ( 3 samples). The research results are presented in table 4.

Table 4 The results of a comparative test of various methods for indicating brucellosis antigen in the test material Material Sample Research Brucellosis antigen detected bacteriological method RNAT RNGA with antibody diagnosticum (PZNIVI) floor. neg. floor. neg. floor. neg. Extract of organs and lymph nodes cr. horn. cattle 7 5 2 6 one 7 - Sheep organ and lymph node extract eleven 10 one 10 one eleven - Brucella infected milk 5 four one four one 5 - Brucella infected tap water 3 3 - 3 3 Total 26 22 four 23 3 26 -

The studies established the specificity and high sensitivity of the antibody erythrocyte diagnosticum to detect the brucellosis pathogen in the material, milk and other environmental objects.

In a comparative test of antibody erythrocyte brucellosis diagnosticum for RNGA with PHAt, it was found that antibody diagnosticum is 22 times more sensitive. In addition, RNGA with antibody diagnosticum is simple to set up, RNAt is a more complex three-component reaction (positive serum + brucellosis antigen + erythrocyte brucellosis antigen).

Brucellosis antibody diagnosticum for RNGA in diagnostic value and speed of obtaining results also surpasses the bacteriological method of research.

LITERATURE

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

Romakhov V.A. 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.

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

Claims (1)

A method of obtaining an erythrocyte antibody diagnosticum for an indirect hemagglutination reaction (RNGA) with the aim of indicating brucella in the material, including the production of formalized ram red blood cells with subsequent sensitization with positive brucellosis serum, characterized in that positive brucellosis serum for sensitization of red blood cellulose is 100-1: , inactivate at 60 ° C for 30 min, formalin erythrocytes sensitize with inactivated serum, based on: t 2 volumes of a 40% suspension of sheep erythrocytes, 1 volume of a 0.43-0.44% solution of freshly prepared amidol, 1.0-1.5 volume of positive blood serum diluted 1: 50-1: 100, and incubated for 28-32 min at 55-56 ° C.