Method of obtaining high-titre antimicrobial serum for estimation of antigen activity of anti-staphylococcal vaccines

FIELD: chemistry.

SUBSTANCE: invention relates to the field of biotechnology. Claimed is a method of obtaining a high-titre antimicrobial serum. Method includes application of strain S.aureus No1991, characterised by immunogenicity ED50=(50-100)×106 microbial cells, weak virulence LD50=(0.5-2.0)×109 microbial cells; its inactivation and drying with dimethylketone by double processing with 3 volumes of dimethylketone. After that, immunisation of animals is carried out: two times subcutaneously and two times intravenously. In conclusion serum isolation is carried out.

EFFECT: invention can be used in medicine for estimation of antigen activity of anti-staphylococcal vaccines.

3 tbl

 

The invention relates to medicine, namely to protivogribkovym vaccines and methods of assessing their antigenic activity. The aim of the invention is to create the most immunogenic non-toxic protivogribkovogo of the drug to obtain vysokotirazhnyh immune sera suitable for evaluation of antigenic activity of staphylococcal vaccines of different antigenic composition at all stages of their preparation, control of the final product and stability during storage.

The advantage of the invention is to standardize methods of control protivotrombovyh products intended for active specific prophylaxis and therapy of staphylococcal infections through the use of serological methods based on the use vysokotirazhnyh antimicrobial protivotrombovyh sera, for which we propose a method of preparing immunogenic, non-toxic and labellling drug.

Known methods for producing antistaphylococcal plasma and serum. The disadvantage of these methods is the induction of antibodies to individual factors of pathogenicity of Staphylococcus aureus and low level of antibodies[2, 3, 4, 9, 10].

Worldwide is the development of staphylococcal vaccines of new generation. Significant efforts in research and to mirceski organizations were focused on obtaining and testing of monovalent vaccines, consisting of capsular polysaccharides (Staphvax), alpha-toxin, other pathogenicity factors [5] Staphylococcus. Study most of these drugs was stopped at the stage of pre-clinical trials. The most well studied vaccine Staphvax, the effectiveness of which in clinical trials has not been confirmed. These data led the authors to the conclusion about the necessity to develop polyvalent staphylococcal vaccines with an optimal set of antigens[6, 7, 8].

In Russia, carried out research on the development of cell-free polyvalent antistaphylococcal vaccines based on water-soluble protein and polysaccharide antigens of the cell wall S.aureus. Preclinical studies have established low toxicity and significant protective activity of this drug [1]. However, even using mnogokubovyh complex schemes of immunization with this vaccine does not provide vysokotirazhnyh sera. At the same time revealed a correlation between antibody titers and survival of rabbits on the model of generalized staphylococcal infection in rabbits (table 1), which determines the possibility of the use of serological methods for the evaluation of immunogenic activity of staphylococcal drugs.

Table 1
Immunogenic activity of dry staphylococcal vaccine in experiments on rabbits (antibody titers and protection on the model of generalized staphylococcal infection)
no experienceNo. seriesThe multiplicity of immunizationImmunizing dose mgGraftedUnvaccinated
Reverse cf. the title at±mSurvived from the number of rabbits taken in the experienceReverse cf. the title at±mSurvived from the number of rabbits taken in the experience
169A2484±19,84/500/5
B2411±2,91/5
27034106±22,3 3/53±0,330/5
702448,4±4,32/5
73323±0,210/5
3763480±5,75/52±0,250/5
41534147±213/56±0,40/5

When developing protivotrombovyh vaccines and in preclinical studies evaluating drugs produced in experiments on animals (mice, rabbits, rare monkeys). These studies are time-consuming. When you research often get different results due to different sensitivity the spine of mice, they are difficult to use on the stages of production, and, as shown by many studies, the protective activity established in mice, has not been confirmed in clinical trials [5]. The claimed method of obtaining immunogenic non-toxic protivogribkovogo preparation for immunization of animals with the aim of obtaining vysokotirazhnyh immune sera will be used to assess immune staph drugs in serological (competitive ELISA and others) and immunochemical (immunoblot, immunodiffusion) reactions.

To achieve this purpose: 1) strain S.aureus 1991, isolated from a patient with pneumonia, characterized by high intra-specific protective activity of the ED50=(20-100)×106microbial cells with weak virulence - LD50>1×109microbial cells; 2) the most gentle method of inactivation of microbial mass dimethylketone; 3) short circuit immunization, consisting of two subcutaneous and two intravenous institutions (table 2).

Table 2.
Comparative data protective and sensitizing activity of preparations derived from different strains of Staphylococcus
Strain Protective activitySensitizing activity
No.LD50, MIC. CL ×109strain to infectmouserabbitsGuinea pigs
survived/total%±msurvived/totalMs. cont. life, days±mpreparation from strainthe ratio p/introductionindex of anaphylactic shock, %±m
60,161/244,0±3,00/86,2±2,561-Crat70±4,8
20030,2-"-4/2020,0±8,9the concentration isthe concentration is20033-Crat75,0±5,8
-"--"-19866/2821±7,6the concentration isthe concentration isthe concentration isthe concentration isthe concentration is
19860,18-"-18/4540±7,22/427,0±7,6the concentration isthe concentration isthe concentration is
19911,0613/2065±106/1020,0±4,519911-Crat27,5±2,9
19911,0198623/4551±7,04/460 (observation period)-"-3-Crat28,5±4,0
Control intact.)-6/td> 0/2500/102,0±0,05Intake.14,2±3,6
-19862/258±5,00/44,0±3,0Intake.12,0±3,2

Selection of antigenic components produced by inactivation and drying microbial mass of cells dimethylketone by 2-fold processing in the amount of 3 volumes relative to the volume of the microbial mass.

An example of the method.

Strain S.aureus 1991 (deposited in gisk named after. L.A. Tarasevich) were cultured on solid agar medium on the basis of pancreatic hydrolysate of casein at 37°C for 16-18 hours. The culture is washed off with distilled water. Possible cultivation in reactors by submerged cultivation for 7-8 hours at a temperature of 37°C or through managed processes of cultivation (waste and topping-up, continuous) polysynthetic environment. For example, the environment of salts KN2RHO4To2NRA4, (NH4)2SO4, MgSO4, sodium citrate, with the addition of glucose. and the yeast EXT the act.

Under cultivation in reactors branch of the microbial biomass from the culture medium produced by centrifugation or separation, it is also possible by microfiltration in a tangential flow.

Inactivation of microbial mass produced by 2-fold processing dimethylketone within 18-24 hours of 3 volume relative to the volume of the microbial mass. After the 2nd treatment get dry microbial mass, standartizeta by weight.

The obtained dry inactivated microbial mass is used to immunize animals (mice, rabbits). High protective activity can significantly reduce the immunization scheme to obtain vysokotirazhnyh sera to 2-3 courses:

year 1 - 2 times subcutaneously at intervals of 3-4 days;

2 year 2 times intraperitoneally with an interval of 3-4 days;

3 the course is carried out additionally under the scheme of the 2nd course only in case of insufficiency of 2 courses (table 3).

Table 3
The titer of the rabbit sera in TPHA in the dynamics of the 3 courses immunization staphylococcal antigen
Antigenic staphylococcal preparation, prepared by the claimed methodNo. of sera of rabbits Reverse antibody titer
before immunizationafter 1 year p/immunizationafter 2 courses/immunizationafter 3 courses/immunization
1234
11625610245120
232102410245120
33251210245120
43212810242560
51625610245120
M±m25,6±3,4435±159,91024±04608±512
P1 and 2<0,05; R2 and 3<0,01; P3 and 4<0,001

Literature

1. Egorova NB, Ephraim V.N., Kurbatova EA, Gruber IM Experimental and clinical and immunological evaluation of acellular staphyococcus vaccine "Stafilov". Journe. microbiol. 2008. No. 6. - P.101-108.

2. Ivanova SP. Method of obtaining immune antistaphylococcal serum. Lab. Case. 1975. No. 5. - 306-308.

3. Mikhailova N.A. and others Experience the vaccine "pipol" for immunization of donors-volunteers. Immunology. - 2006. - V.27. No. 2. - P80-83.

4. Fedorovskaya E.A. and other Immune response of the donor when immunization staphylococcal toxoid. Medical business. 1989. No. 2. - P.70-72.

5. Otto M. Novel targeted immunotherapy approaches for Infection gets. Expert Opin Biol Ther. 2010. - Vol.10. No. 7. P - 1049-1059.

6. Poole-Warren LA, Hallett MD, Hone PW, SH Burden, Farrell PC. Vaccination for prevention of CAPD gets associated infection: results of a prospective multicentre clinical trial. Clin. Nephrol.1991. - Vol.35. No. 5. P.198-206. [PubMed: 1855327]

7. Schaffer AC, Lee J.C. Gets vaccines and immunotherapies. Infect. Dis. Clin. North Am. 2009 - Vol.23. No. 1. 153-71. [PubMed: 19135920]

8. Stranger-Jones Y.K., Bae So, Schneewind O. Vaccine assembly from surface proteins of Staphylococcus aureus. Proc. Natl. Acad. Sci. 2006. - Vol.103. No. 45. - P16942-7. [PubMed: 17075065]

9. Ekstedt R.D. Ann N.Y. Immune response to surface antigens of Staphylococcus aureus and their role in resistance to disease gets. N.Y. Acad. Sci., 1974. - Vol.236. - P.203-220.

10. Richter U., Karsch W.Z. Production of diagnostic antisera for the detection of gets enterotoxins A-e Gesamte. Hyg. 1980. - Vol.26. - N1. - P.53-55.

The method of obtaining vysokotirazhnoj antimicrobial serum for about Enki antigenic activity protivotrombovyh vaccines including the use of S.aureus strain No. 1991, high immunogenicity ED50=(50-100)×106microbial cells, weak virulence LD50=(0.5 to 2.0)×109microbial cells, inactivation of him and drying dimethylketone by 2-fold processing 3 volumes dimethylketone with subsequent immunization twice subcutaneously and intravenously twice and isolation of serum.



 

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