RU2252962C1 - Method for production of brucellosis antigen - Google Patents

Abstract

FIELD: microbiology, immunology, veterinary.

SUBSTANCE: vaccine strain Brucella abortus 104 biovar.1 is cultivated and microbial cells are washed. Then precipitate is resuspended with alkali, preferably 0.5 N NaOH solution, under continuous agitation to produce finished microbial cell concentration of 1x10¹¹ cells in 1 ml. Product is hydrolyzed at 55-57°C under continuous agitation for 10-12 min followed by cooling at 4-6°C for 30 min or more. Suspension is neutralized with 2 N acetic acid and antigen is precipitated with ethanoly preferably in ratio brucellosis antigen/ethanol of 1:10. Method of present invention makes it possible to increase brucellosis antigen yield up to 30 % (calculated as dry microbial mass).

EFFECT: simplified, accelerated and effective method for brucellosis antigen production.

3 cl, 2 tbl, 1 ex

Description

The invention relates to biotechnology, in particular to medicine and veterinary medicine, and can be used for the production of brucellosis diagnosticums.

A known method for producing brucellosis antigen used for the diagnosis of brucellosis, including cultivation of brucella, inactivation of bacterial cells by heating and staining (RU 2133470 C1, 07.20.1999). However, the antigen obtained by this method is used only for the formulation of agglutination reactions of Wright and Hedlson.

There is also known a method of obtaining a single brucellosis antigen for the agglutination reaction (RA), complement fixation reaction (RAC) and long-term complement fixation reaction (RDSK). It includes the cultivation of strain B. abortus 19, concentration and purification in an ultrafiltration unit, as well as inactivation of the antigen in the bioreactor at a temperature of 80 ° C for one hour (RU 2085212 C1, 07.27.1997). This method requires special technical means for its implementation.

There is also known a method for producing brucellosis antigen, including growing brucella, washing microbial cells, processing them with alkali, neutralization with acetic acid, precipitation with acetone and / or ethyl alcohol, drying. The antigen obtained by this method is used to set up an indirect hemagglutination reaction (RNGA), an antibody neutralization reaction (RNAt), and an aggregate-hemagglutination reaction (RAGA) (RU 2086258 C1, 08/10/1997) - the closest analogue.

However, this method of producing brucellosis antigen has disadvantages: a vaccine strain of B. abortus 19 is used, which is unstable, subject to dissociation, as a result of which a low yield of antigen is observed. In addition, the length of the process for obtaining brucellosis antigen is long - from the moment of cultivation, it is 7 days.

The objective of the present invention is to increase the yield of antigen, simplifying the technology, reducing the duration of the process of obtaining antigen.

The technical result - increasing the yield of antigen, simplifying the technology and reducing the duration of the process is achieved by the fact that the method for producing brucellosis antigen involves culturing the vaccine strain Brucella abortus, washing the microbial cells, resuspending them with alkali, followed by neutralization with 2N acetic acid, ethanol precipitation of the target product and drying.

The vaccine strain Brucella abortus 104 biovar 1 is cultivated, registration number 1425 at the Brucella Museum of Live Cultures of the GUNIIEM named after NF Gamalei RAMS, while after processing the microbial cells with alkali, hydrolysis is carried out at a temperature of 55-57 ° C with constant stirring for 10-12 minutes, followed by cooling at a temperature of 4-6 ° C for at least 30 minutes.

The method may be characterized in that the resuspension of microbial cells is carried out with a 0.5 N alkali solution with constant stirring to a final concentration of 1 × 10 ¹¹ microbial cells in 1 ml.

The method can also be characterized in that the target product is precipitated with ethanol with a volume ratio of brucellosis antigen / ethanol equal to 1:10.

The invention is characterized by the use of the vaccine strain B.abortus 104 biovar 1, registration number 1425 in the museum of live cultures of brucella GUNIIEM them. NF Gamalei RAMS, which has high stability, a more complete antigenic structure and abundant growth on solid nutrient media. It is a typical and persistent S-variant; does not form H ₂ S; grows on media with fuchsin and does not grow on media with thionine; agglutinated with anti-abortus serum and not agglutinated with anti-melitensis serum.

To implement the method does not require any special equipment. The strain is cultivated on solid nutrient media in glass mattresses, the hydrolysis of the bacterial suspension is carried out using a water bath and a refrigerator, and the precipitation is carried out by centrifugation.

As experiments showed, the recommended mode of successive heating processes (56 ± 1 ° C) for 10-12 min and cooling (4-6 ° C) for at least 30 min is optimal for complete hydrolysis, accompanied by extraction of antigens from the brucella cell wall .

Example. Brucella vaccine strain B. abortus 104 biovar 1 was grown for 48 hours. Microbial cells were washed with neutral physiological saline (NFR), centrifuged, and the pellet was washed three times with NFR by centrifugation. Next, the pellet was resuspended in a 0.5 N NaOH solution with constant stirring to a final suspension concentration of 1 × 10 ¹¹ microbial cells in 1 ml. Then, the suspension was hydrolyzed at a temperature of 56 ± 1 ° С with constant stirring for 10 min and cooled at a temperature of + 4 ° С for 30 min. Next, the suspension was neutralized with 2 N acetic acid to pH = 7.0, antigen was precipitated with ethanol in a volume ratio of 1:10, the precipitate was separated by centrifugation and lyophilized.

Table 1 shows the comparative characteristics of the parameters of the methods, and Table 2 shows the comparative immunochemical characteristics and the main composition of brucellosis antigens obtained by the patented method and method - the closest analogue.

From the data presented table. 1 shows that the patented method allows to increase the yield of brucellosis antigen by 2 times, and also reduce the duration of the process of obtaining antigen by more than 1.5 times (from 7 days to 4 days).

From table 2 it follows that the method allows to obtain a complete antigen of protein-lipopolysaccharide nature.

From the research results it also follows that the obtained brucellosis antigen can be used not only as an antigen for the production of RNGA, RNAt, ELISA, but also in the latex agglutination reaction (RAL).

Table 1. Comparative characteristics of the proposed method for producing brucellosis antigen and prototype No. p / p Characteristic The proposed method for producing antigen Prototype 1. The yield of antigen in% (on the dry weight of the microbial mass) thirty fifteen 2. The use of producer strain Vaccine strain B. abortus 104 biovar 1 Vaccine strain B. abortus 19 3. Antigen preparation time from the moment of cultivation per day. 4 7 4. The use of brucellosis antigen RNGA, RNAT, IFA. RAL for anti-brucellosis serum of high activity and specificity RNGA, RNAT, RAGA,
to obtain anti-brucellosis serum of high activity and specificity

Table 2. Characterization of brucellosis antigens obtained by the proposed method and the known method (prototype) Brucellosis antigen obtained by the method Serological activity in the credits: Content in% (on dry weight of antigen) ring precipitation reactions Rnga Common sugar Protein Common lipids Nucleic acids Proposed 1; 256000 1: 12800 42 36 fifteen 5 Prototype 1: 128000 1: 12800 38 25 12 3

Claims (3)

1. A method of producing a brucellosis antigen, comprising cultivating a vaccine strain of Brucell abortus, washing microbial cells, resuspending them with alkali, followed by neutralization with 2N acetic acid, ethanol precipitation of the target product, and drying, characterized in that the Brucell abortus vaccine strain is registered at number 14 in the museum of living cultures of brucella GUNIIEM them. N.F. . 2. The method according to claim 1, characterized in that the resuspension of microbial cells is carried out with a 0.5N alkali solution with constant stirring to a final concentration of 1 × 10 ¹¹ mcl in 1 ml. 3. The method according to any one of claims 1 and 2, characterized in that the target product is precipitated with ethanol with a volume ratio of brucellosis antigen: ethanol equal to 1:10.