The method of obtaining diagnostic agglutinins vaccine against brucella in an l-shape

 

(57) Abstract:

The invention relates to the field of veterinary medicine and can be used for diagnosis of the causative agent of brucellosis in an L-shape. The essence of the invention lies in the fact that the serum obtained from the blood of rabbits after conducting dvuhtokovoe hyperimmunization antigen from subculture C. abortus And 206 in an L-shape. The technical result is to obtain highly active and highly specific diagnostic serum against Brucella in an L-shape. 1 PL.

The invention relates to the field of medicine and veterinary medicine, namely to Microbiology and immunology, and can be used for obtaining immune serum used for diagnosis of the causative agent of brucellosis in an L-shape.

It is known that the activity and specificity of immune serum depends on the quality of diagnostic products. Brucellosis polyvalent serum is not agglutinate brutally in a stable L-form and therefore cannot be used for their diagnosis. Therefore, obtaining high-affinity and high-level diagnostic agglutinins vaccine against Brucella in an L-shape is an important task.

A method of obtaining syware is (105M. K. - 3·109M. K.) only after 60-90 days marked mild antibody production (1:10 in RA, 1:40 in RAC and RDSC) [ishchanova R. J., Sementsova C. M., E.E. C. M., Khasanov N. X. // Veterinary medicine, 1986, No. 9, - n-30-32].

It is known that L-forms of Brucella have less pronounced compared to the original bacterial form agglutinability properties, they interacted in agglutination reactions (RA) own L-anticorodal in a dilution of 1:640, while the original culture was agglutinability with S-anticorodal up to a titer of 1:1280. However, anticavity to L-forms were agglutinative and initial bacterial forms in a dilution of 1:320 [Bazikov I. A. fabrication and characterization of L-forms of Brucella and their revertants. Abstract. Diss.... Kida. the honey. of Sciences, Saratov, 1990, - n-16]. The above messages indicate receipt of the sera against Brucella in an L-shape, but with low activity of the antibodies (AT) (titers at 1:10 and 1:640) and specificity, as they react with Brucella in the S-form in the credits AT (1:320), lower than only one breeding than with Brucella in L-form.

Closest to the present invention is a method of obtaining a vaccine against Brucella in an L-shape, including use as an antigen (AG) immunization incdpass is performed using the antigen, prepared from Brucella in an L-shape, inactivated acetone (acetone powders-up). If hyperimmunization rabbits up of Brucella in the L-form are obtained serum, in which the titles at RA was with homologous AG 1:800-1:1600, with a standard Brucella diagnosticum 1:400-1:800 [Dranovsky E. A., Tolmachev, T. A. Rostovtsev N. A. // Ukr. microbiol., 1981. No. 7. - C. 33]. However, this method does not allow to obtain serum against Brucella in an L-shape with high activity and specificity. Shown in prototype serum against Brucella in an L-shape has a low reactivity with Brucella in L-form (titres AT 1:800-1:1600) and specificity, as it reacts with the standard Brucella diagnosticum (S-form) in the credits AT less than only one dilution (1:400-1:800), than with L-antigen.

The aim of the invention is to obtain highly active and highly specific diagnostic agglutinins vaccine against Brucella in L-form.

This objective is achieved in that exercise dvuhlitrovuyu hyperimmunization rabbits AG from subculture C. abortus And 206 in an L-shape on the original scheme, to keep the blood from which it is prepared serum.

Comparative analysis of the prototype showed that the proposed technical R is of diagnostic serum used dvuhlitrovuyu scheme hyperimmunization inaktivirovannye AG, made from subculture C. abortus And-206 to L-form.

In the first cycle before immunization of rabbits in the pads of the hind paw injected with 0.5 to 0.6 ml complete adjuvant's adjuvant (PAF), then after 6-7 days injected AG combined in the popliteal lymph nodes and intramuscularly (suspension of AG with PAF). After another 3-4 days repeat injections, but in the front paws and intramuscularly. On 29-31 days from the beginning of immunization, before the second cycle, prepare the complex antigen-antibody (AG-AB). After 30-32 days to have a second cycle, which rabbits subjected to immunization in combination: a complex of antigen-antibody injected, and the suspension of AG with PAF - intramuscularly. After 3-4 days of injections of a complex of AG-AB and AG repeat. After 7-9 days after the last immunization, the animals will Krasouskaya and receive diagnostic agglutinating serum against Brucella in L-form.

Thus, the proposed solution meets the criteria of the invention of “novelty.”

The analysis of patent and scientific literature showed that the proposed method differs not only from the prototype, but also on other technical solutions in this and related fields. So, the authors have not found a way poluchenno, the proposed modes allow you to achieve this goal is to obtain a highly active and highly specific diagnostic agglutinating serum against Brucella in an L-shape. This method does not require special equipment and reagents and can be used in the production of this serum in the laboratory.

The proposed diagnostic agglutinating serum can be used in medicine and veterinary medicine for the diagnosis of modified forms of Brucella, in particular L-forms. Thus, this method meets the criteria of “inventive step” and “industrial applicability”.

The method is as follows:

Take inactivated subculture C. abortus And 206 in an L-shape and are hyperimmunization rabbits under the scheme, including two cycles.

First cycle: the rabbits in the pads of the hind paw injected with 0.5 to 0.6 ml of PAF. After 6-7 days the animals are subjected to immunization in the popliteal lymph nodes of the rear paws of 0.3-0.5 ml of a suspension of AG in buffered saline solution pH 7,2-7,4 (SFR) at a dose of 4·108-5·108microbial cells (M. K.) and intramuscularly at a dose of 4·108-5·108M. K. in a volume of 1.0 ml containing 0.5 ml of suspension of AG and 0.5 ml·109M. K. and intramuscularly 109-2·109M. K. in a volume of 1.0 ml (suspension of AG-PAF 0.5 ml).

On 29-30 days from the beginning of immunization prepare the immune complex of antigen-antibody, for what animals are immune serum, which is poured in two bottles of 1.0 ml In each tube add 1 ml of suspension of AG in the corresponding immunizing dose.

The second cycle of immunization is carried out through 30-32 days. The complex of antigen-antibody administered intravenously at the rate 2.5·109-3·109M. K., and intramuscular injection of a suspension of AG based on 2·109-3·109M. K. in the amount of 0.25-0.5 ml 0.5 ml PAF. After 3-4 days, make repeated intravenous injections of the complex AG-AB 3·109-4·109M. K. and intramuscular injection of a suspension of AG - 2·109-3·109M. K. in 0.25-0.5 ml SPR with 0.5 ml of PAF.

After 7-9 days after the last immunization, the animals spend bloodletting and get the serum in a standard way.

Example 1.

Take an inactivated antigen of subcultures C. abortus And 206 in an L-shape and are hyperimmunization according to the following scheme.

First cycle: the rabbits in the pads of the hind paw injected with 0.6 ml of PAF. After 7 days the animals are subjected to immunization in the popliteal lymph nodes rear l is Adam 0.5 ml of a suspension of AG and 0.5 ml of PAF. After 4 days of injection of AG carried out in pads front paws in a dose of 2·109M. K. in a volume of 0.6 ml SPR and intramuscularly 2·109M. K. in a volume of 1.0 ml (suspension of AG-PAF 0.5 ml).

On the 31st day from the beginning of the immunization prepare the immune complex of antigen-antibody, for what animals are immune serum, which is poured in two bottles of 1.0 ml In the first test tube add 1.0 ml of a suspension of AG at a dose of 3·109M. K., second 1.0 ml suspension of AG at a dose of 4·109M. K.

The second cycle immunization spend 32 days. The complex of antigen-antibody administered intravenously at the rate of 3·109M. K., and intramuscularly injected with 0.5 ml of a suspension of AG at a dose of 3·109M. K. with 0.5 ml of PAF. After 4 days, make repeated intravenous injections of the complex of antigen-antibody containing 4·109M. K., and intramuscularly injected with 0.5 ml of a suspension of AG (3·109M. K.) with 0.5 ml of PAF.

After 9 days after the last immunization, the animals will Krasouskaya and the standard method of preparing diagnostic agglutinating serum against Brucella in an L-shape. The resulting serum is a specific title AT 1:1600-1:3200.

Example 2.

Take an inactivated antigen of subcultures C. abortus And 206 in an L-shape and are hyperimmunization of sleh subjected to immunization in the popliteal lymph nodes of the rear paws of 0.3 ml of a suspension of AG at a dose of 4·108microbial cells (M. K.) and intramuscularly at a dose of 4·108M. K. in a volume of 1.0 ml containing 0.5 ml of antigen suspension and 0.5 ml of PAF. After 3 days, the injection of suspensions of AG carried out in pads front paws in a volume of 0.5 ml at a dose of 109M. K. and intramuscularly 109M. K. in a volume of 1.0 ml (suspension of AG-PAF 0.5 ml).

On the 29th day from the beginning of the immunization prepare the immune complex of antigen-antibody, for what animals are immune serum, which is poured in two bottles of 1.0 ml In the first test tube add 1.0 ml of a suspension of AG in the dose of 2.5·109M. K., second 1.0 ml suspension of AG at a dose of 3·109M. K.

The second cycle immunization carried out in 30 days. The complex of antigen-antibody administered intravenously at the rate 2.5·109M. K., and intramuscularly suspension of AG 2·109M. K. 0.25 ml with 0.5 ml of PAF. After 3 days, make repeated intravenous injections of the complex of antigen-antibody at a dose of 3·109M. K. and intramuscular suspension of AG at a dose of 2·109M. K. in a volume of 0.25 ml with 0.5 ml of PAF.

Through 7 days after the last immunization, the animals will Krasouskaya and the standard method of preparing diagnostic agglutinating serum against Brucella in an L-shape. The resulting serum is a specific title AT 1:1600-1:3200.

In addition, the use of serum, obtained by the proposed method allows to detect soluble antigens of Brucella in the L-form in the reaction, immunodiffusion in gel formation of one or two bands of precipitation and does not form any with antigens of Brucella S-form. Brucellosis polyvalent serum in the reaction, immunodiffusion in gel with Brucella antigens in S-shape forms a 2-3 or more bands of precipitation.

The proposed method for the rabbit hyperimmune serum against Brucella in the L-form is more efficient because it involves the immunization of animals inaktivirovannye antigen that does not require special measures for the protection of operating personnel.

Compared with the prototype of this method is, first and foremost those that apply dvuhlitrovuyu hyperimmunization on the original scheme, allowing you to get more specific and more active Syv what about this, compared with the prototype, where the antibody titers with standard Brucella diagnosticum (S-form) was 1:400-1:800, RA proposed L-serum is not registered positive reactions with Brucella in the S - and R - forms, indicating the absence of the necessary adsorption for removal of antibodies against S - and R-antigens of Brucella. The high specificity indicates the absence of interaction diagnostic serum against Brucella in an L-shape with some microorganisms, with several common antigens.

The method of obtaining diagnostic agglutinins vaccine against Brucella in L-form, including immunization of animals with the antigen of subcultures C. abortus in an L-shape, bleeding the animals and obtaining serum, characterized in that hyperimmunization spend antigen obtained from subcultures C. abortus And 206, two cycles as follows: in the first cycle, the rabbits in the pads of the hind paw injected with 0.5 to 0.6 ml complete adjuvant's adjuvant, after 6-7 days enter suspension of antigen in combination: in the popliteal lymph nodes of the rear legs at a dose of 4-10 - 5-10 M. K., and intramuscularly 4·108- 5·108M. K. with complete adjuvant's adjuvant, after 3-4 days of injection powa then through 29-31 days from the beginning of the immunization, the animals spend bloodletting and get the immune serum, to obtain a complex of the antigen - antibody type antigen in a 1:1 ratio in immunizing doses of 2.5·109- 3·109M. K., and 3·109- 4·109M. K. through 30-32 days to have a second cycle of immunization, which includes the introduction of complex antigen - antibody intravenously at a dose of 2.5·109- 3·109M. K., and suspension of the antigen with complete adjuvant's adjuvant intramuscularly at a dose of 2·109- 3·109M. K. after 3-4 days of injections of a complex of antigen - antibody and the antigen suspension is repeated similarly in doses of 3·109- 4·109M. K., and 2·109- 3·109M. K. accordingly, to obtain the serum of the animals will Krasouskaya in 7-9 days.



 

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