RU2624871C1 - Method of obtaining pneumococcal hyperimmune sera - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: rabbit hyperimmunization is carried out by antigen, which is an antigen of plasmid-containing pneumococcus strain, inactivated with formalin. Vaccine immunization is performed in anauricular vein, wherein rabbit immunization with the mentioned antigen is carried out at an interval between each injection, then immunogenic properties are determined in producer, blood is collected, followed by separation and conservation of the resulting serum. In order to produce vaccine of one series, 16-hour culture grown on semi-synthetic nutritional medium is used, which cells are separated by centrifugation. Sludge is resuspended in physiological solution and is inactivated by warming-up, then cells are washed off with physiological solution containing 0.25% of formaldehyde, and microbial suspension corresponding to 4×10⁹ mcr.kl./ml is prepared, wherein pneumococcal microbial cells are washed off before immunization again. Vaccine immunization is carried out 3 times a week for 4 weeks: the first week at doses of 1×10⁸ mcr.kl./ml, the second week - 5×10⁸ mcr.kl./ml, the third week - 1×10⁹ mcr.kl./ml and the fourth week - 2×10⁹ mcr.kl./ml, in the fifth week blood is taken, serum is obtained and its activity is checked in the agglutination reaction (AR), which titer must be in the range of 1:3200-1:6400.

EFFECT: use of this method enables to increase sera specific activity by using vaccines from microbial cells of each of the pneumococcal serotype and rabbit immunization scheme in the preparation.

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Description

The invention relates to medical immunology and may find application for the production of highly active, specific hyperimmune sera [A61K 39/40, C07K 16/12].

Patent RU 2203090 is known for a Method for the production of diagnostic serum with agglutinins for virulent yersinia (SVI) by hyperimmunization of rabbits with plasmid-containing (pYV +) strain Yersinia enterocolitica serovar OZ and subsequent adsorption with plasmid-free (pYV-) variants of rabies, which is different for according to the scheme: 0.09-0.11 ml three times with an interval of 2-3 days and after 7-8 days - 2 more injections with an interval of 2-3 days, and microbial masses of reference strains GISK 189 and GISK 207 are used as adsorbents.

The disadvantage of this solution is tolerance to the CPS; in addition, a serum adsorption step is required. The use of phenol can destroy the inner lining of the rabbit veins and cause thrombosis, which limits the time and amount of serum obtained.

In contrast to this solution, in the claimed invention, serum can be obtained for a long time, repeating the immunization schedule. In our case, the goal is achieved by immunization with whole microbial cells washed from excess polysaccharide to avoid tolerance to CPS; in addition, there is no stage of adsorption of serum, which simplifies the scheme.

Known patent RU 2135210 for a Method for the production of diagnostic serum, including multiple immunization of animal producers with avirulent antigenic material in conjunction with immunomodulators, blood sampling and separation of serum, characterized in that thymalin and cyclophosphamide are used as immunomodulators, immunization is carried out five times, antigenic material is administered immunomodulator thymalin - intramuscularly simultaneously with antigen injection, while cyclophosphamide is administered once with a third injection.

The disadvantage of this solution is the use of immunomodulators and immunosuppressants, which complicates and increases the cost of obtaining serum. In addition, the use of an immunomodulator and an immunosuppressor “depletes” the immune system, which leads to the rejection of production rabbits.

The claimed invention provides immunization with whole microbial cells. The injected vaccine allows you to get specific, highly active serum, without the use of immunomodulators and immunosuppressants.

The closest to the essence and purpose of the claimed method is a method for producing diagnostic agglutinating serum for pathogenic strains of Yersinia (patent RU 2358764, A61K 39/40 from 06/20/2009). The method involves hyperimmunization of rabbits with an antigen, which is a 0.4-0.6% formalin inactivated plasmid-containing (pYV +) antigen of the Yersinia enterocolitica My 03R strain, in the ear cranial vein. Immunization of rabbits with the indicated antigen is carried out four times in doses: 290-310 million cells / ml, 490-510 million cells / ml, 0.99-1.1 billion cells / ml and 1.9-2.1 billion. cells / ml, respectively, with an interval between each injection of 6-7 days. Next, immunogenic properties are determined from the producer, then blood is taken, followed by separation and preservation of the obtained serum. The method provides high-quality agglutinating serum used for the diagnosis of yersiniosis in animals.

The disadvantage of the prototype is not high enough specific activity of the sera.

The objective of the invention is to increase the specific activity of diagnostic sera, simplifying and reducing the complexity of the process of obtaining them with a high yield of the target product.

The technical result of the claimed invention is to improve the quality of serum.

The specified technical result is achieved due to the fact that the claimed method of producing pneumococcal hyperimmune sera, characterized by hyperimmunization of rabbits with an antigen, which is a formalin-inactivated antigen of a plasmid-containing strain of pneumococcus, vaccination is carried out in the ear vein, and rabbits are immunized with each antigen by injection, with an interval between immunogenic properties are determined from the producer, blood is taken, followed by separation and canning irradiated serum, characterized in that to obtain the vaccine of one series, a 16-hour culture grown on a semi-synthetic nutrient medium is used, the cells of which are separated by centrifugation, the precipitate is resuspended in physiological saline and inactivated by heating, then the cells are washed with physiological saline containing 0.25% formalin and preparing a microbial slurry corresponding to 4 × ¹⁰ September mkr.kl. / ml, and the microbial cells before immunization pneumococci again washed, immunization is carried out 3 times a week t chenie 4 weeks: the first week at doses of 1 × ¹⁰ August mkr.kl. / ml, the second - 5 × ¹⁰ August mkr.kl. / ml, and the third - 1 × ¹⁰ September mkr.kl. / ml, and the fourth - 2 × 10 ⁹ μl / ml, blood is taken in the fifth week, serum is obtained and its activity is checked in the agglutination reaction (RA), the titer of which should be in the range 1: 3200-1: 6400.

Since the immunizing dose administered per week is large and with a single administration can cause the death of animals, it is necessary to extend the administration for a week. In the interval between immunizations, the antigen is partially eliminated.

It is believed that a week after immunization, antibodies are formed in the quantity we need.

The technical result of improving the quality of serum is achieved through the use of changes in the preparation of vaccines from the microbial cells of each of the pneumococcal serotypes and the rabbit immunization schedule.

The implementation of the invention

The method is implemented by means of the fact that production rabbits are immunized with an inactivated microbial culture according to the scheme shown in the table:

To obtain a vaccine of one series, a 16-hour culture grown on a semi-synthetic nutrient medium is used. Cells are separated by centrifugation. The precipitate was resuspended in physiological saline and inactivated by heating. Cells are washed with physiological saline containing 0.25% formalin and a microbial suspension is prepared corresponding to 4 × 10 ⁹ μl / ml. To stabilize and intensively produce antibodies of limited heterogeneity, the microbial cells of pneumococci are washed off again before immunization, in order to avoid the development of tolerance under the influence of a free polysaccharide.

If the serums during the test bloodletting contain a large amount of antibodies, then the bloodletting continues for the next 2-3 weeks, and then take a 4-week break. After this, the immunization is repeated, introducing 2 × 10 ⁹ μl. in ml in the first week 3 times and in the next 4 weeks - 2 times. Serum activity is determined in an agglutination reaction. The title should be 1: 3200-1: 6400.

In the patent and scientific and technical literature, technical solutions are not known containing the modes of obtaining hyperimmune sera to pneumococcus strains belonging to different serotypes, similar to the claimed one, i.e. the proposal meets the criterion of "novelty." All modes of the method are feasible in laboratory and industrial conditions, aimed at solving a real technical problem, i.e. the proposal is “industrially applicable”.

The method is illustrated in the following examples.

Example 1

Get hyperimmune serum to the pneumococcal strain of serotype 19A as follows. 1500 ml of a 16-hour culture was centrifuged. The microbial cell pellet was resuspended in 30 ml of saline and inactivated by heating at 56 ° C. Cells were washed three times with physiological saline containing 0.25% formalin and a microbial suspension was prepared corresponding to 4 × 10 ⁹ μl / ml. Before immunization, microbial cells were washed again with saline.

Immunization of rabbits with the vaccine was carried out in the ear vein 3 times a week, for 4 weeks at doses of 1 × 10 ⁸ , 5 × 10 ⁸ , 1 × 10 ⁹ and 2 × 10 ⁹ μl / ml. In the fifth week, blood was taken from rabbits, serum was obtained and its activity in RA was checked. The average titer of antibodies in the obtained blood serum to S. pneumoniae serotype 19A in RA was 1: 6400.

Example 2

Get hyperimmune serum to the pneumococcal strain of serotype 3 as follows. 1000 ml of a 16-hour culture was centrifuged. The microbial cell pellet was resuspended in 20 ml of physiological saline and inactivated by heating at 56 ° C. Cells were washed three times with physiological saline containing 0.25% formalin and a microbial suspension was prepared corresponding to 4 × 10 ⁹ μl / ml. Before immunization, microbial cells were washed again with saline.

Immunization of rabbits with the vaccine was carried out in the ear vein 3 times a week, for 4 weeks at doses of 1 × 10 ⁸ , 5 × 10 ⁸ , 1 × 10 ⁹ and 2 × 10 ⁹ μl / ml. In the fifth week, rabbits were bled three times, serum was obtained and its activity in RA was checked. The average titer of antibodies in the obtained blood serum to S. pneumoniae serotype 3 in RA was 1: 3200.

Claims (1)

A method of producing pneumococcal hyperimmune sera, characterized by hyperimmunization of rabbits with an antigen, which is a formalin inactivated antigen of a plasmid-containing pneumococcus strain, vaccination is carried out in the ear vein, and rabbits are immunized with the indicated antigen with an interval between each injection, then the producer receives blood from the producer separating and preserving the obtained serum, characterized in that to obtain a vaccine one the series use a 16-hour culture grown on a semi-synthetic nutrient medium, the cells of which are separated by centrifugation, the precipitate is resuspended in physiological saline and inactivated by heating, then the cells are washed with physiological saline containing 0.25% formalin, and a microbial suspension corresponding to 4 × 10 ⁹ is prepared μl.cl./ml, moreover, the microbial cells of pneumococci are washed again before immunization, vaccination is carried out 3 times a week for 4 weeks: the first week at doses of 1 × 10 ⁸ μl.cl / ml, the second - 5 × 10 ⁸ mc l / ml, the third - 1 × 10 ⁹ μl / ml and the fourth - 2 × 10 ⁹ μl / ml, at the fifth week take blood, get serum and check its activity in the agglutination reaction (RA), the title of which should be within 1: 3200-1: 6400.