RU2283498C1 - Method for obtaining erythrocytic antigen for reaction of indirect hemagglutination in case of brucellosis - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: the present innovation deals with preparing formalinized erythrocytes followed by their sensitizing with sensitin and being different by the fact that before treating with sensitin formalinized erythrocytes should be pre-treated with sodium dodecylsulfate in 1%-concentration at 50-60°C for 30 min; sensitization is carried out with sensitin obtained due to growing bacterial mass of Brucella followed by washing it out with hypertonic sodium chloride solution, inactivating, extracting and separating sensitin due to centrifuging. The innovation enables to obtain diagnostic preparation of high efficiency for detecting brucellosis in animals.

EFFECT: higher efficiency.

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Description

The invention relates to the field of veterinary medicine, in particular to the preparation of diagnostic preparations, and can be used for serological diagnosis of animal brucellosis.

The known "Method for producing brucellosis erythrocyte diagnosticum" by disintegrating brucella with ultrasound followed by antigen release and sensitization of formalized red blood cells, with the aim of increasing the activity of the diagnosticum, disintegration is carried out at pH 8.0-9.0 and erythrocyte sensitization is carried out at 70-72 ° C 5-10 minutes (AS No. 784879, 1980, Kazakh Scientific Research Veterinary Institute; Karaganda Scientific Research Veterinary Station of the Ministry of Agriculture of the Kazakh SSR; av tori Ivanov N.P., Belchenko V.B.).

The disadvantage of this method is that during a serological study in the RNGA using a diagnosticum manufactured by the specified method, animals from farms that are dysfunctional for brucellosis or artificially infected with brucellosis, it is not possible to establish a diagnosis of this disease in some patients with brucellosis, despite the fact that they have specific hemagglutinins circulating in the blood serum, which are found in RNGA when using a more active erythrocyte antigen (see table 3).

The aim of the present invention is to develop a method for producing an erythrocyte antigen for an indirect hemagglutination reaction (RNGA), which is highly active and makes it possible to detect brucellosis-infected animals most fully and at an earlier time after infection.

The goal is achieved in that:

1. Formalized red blood cells to increase the adsorption properties of their receptors are pretreated before sensitization with a new detergent - sodium dodecyl sulfate in the optimal mode - in 1% concentration at 50-60 ° C for 30 minutes.

2. In order to determine the optimal dose of sensitin required for sensitization of red blood cells and to obtain a standard antigen for RNGA, the national biological standard is used - a standard sample of anti-Brucella abortus serum.

The proposed method is as follows.

The three-day agar culture of brucella strain 19 is washed off with a 12% sodium chloride solution, filtered through four layers of gauze, and the concentration of brucella is adjusted to 80-100 billion microbial cells in 1 ml. The pH is adjusted to 8.0-9.0 and autoclaved at 1 atmosphere (120 ° C) for 20-30 minutes. A suspension of brucella is cooled to a temperature of 18-25 ° C and subjected to centrifugation at 8-10 thousand revolutions per minute. The extract obtained, which is a brucella lipopolysaccharide-protein complex (sensitan), is used to sensitize formalized red blood cells. Prior to sensitization, formalin erythrocytes are treated with a detergent - sodium dodecyl sulfate at a 1% concentration at a temperature of 50-60 ° C for 30 minutes.

Sensitization of formalin erythrocytes after treatment with sodium dodecyl sulfate is carried out by the optimal dose of sensitin, which is determined by titration using a standard sample of abortus anti-brucella serum.

The specificity and activity of the finished erythrocyte antigen is checked by examining negative serum in a rnga and a standard sample of abortus anti-brucella serum.

An example of determining the optimal conditions for processing formalin erythrocytes with sodium dodecyl sulfate and sensitizing them with sensitin (lipopolysaccharide-protein complex).

The determination of the optimal concentration of sodium dodecyl sulfate required to increase the adsorption properties of erythrocytes and to obtain a specific and active erythrocyte antigen for RNGA is carried out by processing 10% suspension of formalin erythrocytes with different concentrations (1.0%; 1.5,%) of sodium dodecyl sulfate and checking the activity and specificity in RNA of antigen obtained by sensitization of red blood cells treated with different concentrations of the specified detergent.

The optimal sensitizing dose of sensitin necessary to obtain a standard antigen for RNHA with optimal activity is determined by sensitizing formalin erythrocytes (pre-treated with sodium dodecyl sulfate) with increasing doses of sensitin and testing the serological activity of sensitized erythrocytes (antigen) in RNGA using a standard sample. To sensitize red blood cells, 0.5 ml is added to 1 ml of their 5% suspension; 1.0; 1.5; 2.0 ml of sensitin. The results of testing the activity and specificity of the antigen obtained by processing red blood cells with different concentrations of sodium dodecyl sulfate and sensitized with different doses of sensitin are given in table 1.

Table 1
The results of titration of sensitin and verification of the activity of erythrocyte antigen in RNGA Amount of sensitin per 1 ml of 5-% suspension of red blood cells RNGA Titles with a standard sample of anti-brucella abortus serum with negative serum with saline 1: 200 1: 400 1: 800 1: 1600 1: 3200 1: 6400 1:50 1: 100 1: 200 1: 400 one 2 3 four 5 6 7 8 9 10 eleven 12 but. with antigen made without treating red blood cells with sodium dodecyl sulfate 1,0 + - - - - - - - - - - 1,5 ++ + - - - - - - - - - 2.0 ++ ++ + - - - - - - - - 2,5 +++ ++ + - - - - - - - - 3.0 +++ ++ ++ + - - - - - - - b. with an antigen made by treating red blood cells with a 1% concentration of sodium dodecyl sulfate 1,0 ++++ ++++ ++++ ++++ + - - - - - - 1,5 ++++ ++++ ++++ ++++ ++++ - - - - - - 2.0 ++++ ++++ ++++ ++++ ++++ + - - - - - 2,5 ++++ ++++ ++++ ++++ ++++ ++ - - - - - 3.0 ++++ ++++ ++++ ++++ ++++ +++ - - - - - at. with an antigen made by treating red blood cells with a 1.5% concentration of sodium dodecyl sulfate 1,0 ++++ ++++ ++++ ++++ + - - - - - - 1,5 ++++ ++++ ++++ ++++ +++ - - - - - - 2.0 ++++ ++++ ++++ ++++ ++++ + - - - - - 2,5 ++++ ++++ ++++ ++++ ++++ ++ + + + + - 3.0 ++++ ++++ ++++ ++++ ++++ +++ ++++ ++++ + + - Note: the sign is negative.

From the data shown in table 1, it is seen that the antigen made by sensitizing red blood cells not treated with sodium dodecyl sulfate has low activity. The treatment of red blood cells with sodium dodecyl sulfate increases the activity of the red blood cell antigen. The most optimal for the treatment of red blood cells is a 1% concentration of sodium dodecyl sulfate, which allows one to obtain an erythrocyte antigen with a sufficiently high activity and not giving non-specific reactions with negative serum and saline. The smallest dose of sensitin, which allows one to obtain an antigen with a sufficiently high activity, is 1.0-1.5 ml of it per 1 ml of 5% suspension of formalin erythrocytes pretreated with a detergent.

Therefore, processing of red blood cells with a 1.0% concentration of sodium dodecyl sulfate and sensitizing them, at the rate of 1-1.5 ml of sensitin per 1 ml of 5% suspension of red blood cells, allows you to obtain a specific and more active (compared to analogue) brucellosis erythrocyte antigen for RNGA (tables 2 and 3).

table 2 The results of the test for the specificity of erythrocyte antigens (diagnosticums) manufactured by various methods in RNGA №№ Serum, blood tested Tested in rnga serum Research result with antigen Prikasp. zonal NIVI with diagnosticum of Kazakh NIVI (analogue) one. healthy cattle brucellosis fifty - - 2. healthy brucellosis sheep 60 - - 3. healthy brucellosis pigs 3 - - four. cattle tuberculosis patient 7 - - 5. cattle leukemia 10 - - 6. cattle leptospirosis 8 - - 7. patients with campylobacteriosis of sheep 5 - - 8. tularemia 2 - - 9. paratuberculosis patient cr. horn. cattle one - - 10. sick B.ovis rams 3 - - Total investigated: 149 - - Note: (-) - negative result.

Table 3 The results of a comparative test of the activity of brucellosis erythrocyte antigens (diagnosticums) for RNGA made in various ways Animal species Blood serum tested Indicators Research result with antigen of the Caspian zonal NIVI with the diarnosticum of the Kazakh NIVI (analogue) Croup. horn. livestock of dysfunctional brucellosis farms 54 Received positive RNGA 32 26 % 59.2 48.1 The average titer of RNGA 1: 409 1: 390 Small horn. livestock of dysfunctional brucellosis farms 10 Received positive RNGA 6 - % 60 - The average titer of RNGA 1: 158 - Pigs artificially infected with brucellosis 10 Received positive RNGA 10 9 % one hundred 90 The average titer of RNGA 1: 425 1: 200

Claims (1)

A method of producing an erythrocyte antigen for an indirect hemagglutination reaction during brucellosis, comprising preparing formalin erythrocytes followed by sensitization with sensitin, characterized in that prior to sensitin treatment, formalized red blood cells are pretreated with sodium dodecyl sulfate at a concentration of 1% at 30-60-60 ° C for , sensitization is carried out by sensitin obtained by growing the bacterial mass of brucella, washing it off with a hypertonic solution of sodium chloride, inactivation, ext sensitin line of response and separation by centrifugation.