Method for producing diagnostic anthrax allergen

FIELD: medicine.

SUBSTANCE: method provides recovery of a diagnostic anthrax allergen directly from a concentrated suspension of vegetative cells of a vaccine strain Bac. anthracis STI-1. The strain is grown on a nutrient medium based on a muriatic hydrolyzate of a fish flour by deep cultivation. The produced bacterial mass is washed in a separator to produce the concentrated suspension of vegetative cells. An allergenic protein fraction is recovered by alkaline hydrolysis, and the prepared hydrolyzate is fractioned. A protein allergen fraction is recovered from a supernatant of the recovered fraction by fractioning with an acetic acid solution; further the precipitation containing an end product is dissolved, dialyzed to produce a purified anthrax protein allergen. The preparation is presented in liquid and lyophilised forms.

EFFECT: method allows higher safety of producing the preparation and reduced length of a technological process, with preserving long-storage activity, effectiveness both in evaluating the anti-anthrax immunity stress, and diagnosing the infection.

4 cl, 4 tbl

The invention relates to the production technology of diagnostic drug that is required for human and animal health when assessing the strength protivovirusnogo immunity and diagnosis of infection in persons to work with anthrax.

Analogs of the invention are dry anthrax diagnostic allergen created FGI "48 Central research Institute of the Ministry of defense of the Russian Federation" /1/, and liquid drug anthracen developed Moldovan IMG /2/.

The purpose of the invention: apparatus and process lines for the production of anthrax vaccine /3/ to develop a method of obtaining diagnostic anthrax allergen, which is not inferior in their basic properties counterparts, and in the quantity necessary for practical use.

This goal was achieved by the selection of diagnostic anthrax allergen directly from a concentrated suspension of vegetative cells vaccine strain of Bacillus anthracis STI-1, excluding the stage of obtaining dried acetone cells, which included the processing of a concentrated suspension of vegetative cells tenfold volume of chilled acetone with subsequent drying at room temperature for 26 hrs. /1/. It is known that acetone Rel is referring to the toxic they are flammable and explosive substances, which are increased requirements /4/.

The invention

1. Preparation of seed culture production strain Bacillus anthracis STI-1 exercise from the mother culture reference vaccine strain derived from gisk named after. Laurasia. The process of obtaining seed culture includes preparation of vegetative culture in liquid nutrient medium (HRSA), obtaining spore culture on solid nutrient medium (PPP), washing, separation and filling into capsules.

2. Preparation ZhPS on the basis of 1% hydrochloric acid hydrolysate of fish meal. ZhPS consists of the following components: 1% hydrochloric acid hydrolyzate, fish meal, corn extract and purified water. After filtering ZhPS contains Aminova nitrogen from 0.39 to 0.41 g/DM³and has a pH from 7.8 to 8.0.

3. Preparation of vegetative culture of the strain Bacillus anthracis STI-1 in the reactor bear of 0.1. The process is a welcome and sterilization ZhPS in the reactor, the introduction of additives (glucose, calcium chloride and manganese sulfate), antifoam and seed culture is based from 300 to 500 thousand dispute on 1 cm³environment. ZhPS after the introduction of additives must meet the following requirements:

the content of total nitrogen, g/DM³from 1.2 to 1.6;

content Aminova nitrogen, g/DM³from 0.3 to 0.5;

the content of reducing substances, g/DM³- from 3.9 to 4.1;

chloride (based on NaCl), g/DM³- from 1.6 to 3.0;

the pH from 7.5 to 7.9.

Growing vegetative cells in the reactor bear-0,1 carried out at a temperature from 32 to 33°C and the pressure in the cavity of the reactor from 0.02 to 0.03 MPa with a constant supply of sterile air for aeration through the annular barbaterom with a flow rate from 13.8 to 14.2 DM³/min and a stirring rotation speed of the stirrer 8.3 sec^-1(500 rpm). The cultivation process is conducted for 13-14 hours.

4. Preparation of washed from HRSA concentrated suspension of vegetative cells carry on the separator VDR-3MB dual shaded cells purified water when serving in mesterolone space drum separator native vegetative culture at speeds from 30 to 40 DM³/PM

5. To obtain protein allergens fraction used method of alkaline hydrolysis. The hydrolysis is carried out with 1% solution of potassium hydroxide at the rate of 100 cm³alkali (12±1) g of a concentrated suspension of vegetative cells during 25-29 hours at a temperature of from 20 to 24°C with periodic mixing 3-4 hours within 5 minutes the resulting hydrolysate fractionary on high-speed refrigerated centrifuge with rotor speed 12000 rpm and a temperature of from 3 to 5°C. the Allocation of protein allergens f is the action performed by the fractionation of the supernatant liquid 50% solution of acetic acid at the rate of 1 volume of acid to 4 volumes of liquid and incubated for 4-6 hours at a temperature of from 3 to 5°C. The deposition of protein allergens faction carried out using centrifugation. The obtained protein allergen fraction dissolved in sterile 0.9 percent aqueous sodium chloride solution, deleteroute in sleeves viscose tube and lyophilizers.

The exception at this stage the selection of the allergen being received esetonlinescanner cells /1/ helped to reduce the duration of the technological process on 26 h, to reduce its complexity and increase security.

6. To get ready the liquid form of anthrax diagnostic allergen lyophilized protein allergen fraction dissolved in borate buffer solution with a pH from 7.2 to 7.4 rate of 20 µg protein in 0.1 cm³(the protein concentration determined by the method of Lowry), the solution autoclave at a temperature of from 108 to 112°C for 18-22 minutes the resulting solution to remove extraneous microflora and Department of thermolabile proteins filtered through a membrane "Vladipor" with a pore diameter of 0.22 μm. Sterile solution is a liquid form of anthrax diagnostic allergen - poured into 1.0 ml in ampoules and sealed.

7. To obtain the dry form of anthrax diagnostic allergen sterile solution is poured into 1.0 ml in ampoules and freeze-dried plants LZ-45.27 or AXS. Temperature drying regulate the supply is of Ala to material from minus 32°C to + 28°C. The total duration of the process of freeze drying is from 20 to 24 hours.

8. The assessment of specific activity of liquid and dry forms of the obtained preparation was carried out on a single immunized with anthrax vaccine STI-1 cavies index of the intensity of the reaction (table 1) /5/. Drug comparison was dry anthrax diagnostic allergen, made from dried acetone cells /1/.

As a result of the research it was found that obtained by our proposed method the drug in a diagnostic dose of 20 µg protein in 0.1 cm³allows assessment protivovirusnogo immunity in vaccinated animals. This positive reaction was observed in all experiments. Index of the intensity of the reaction and the percentage of positive samples of all the drugs did not differ among themselves.

9. With the help of a product obtained by the proposed method was evaluated the possibility of diagnosis of anthrax infection (table 2).

For this purpose, Guinea pigs subcutaneously infected with spores of strain 71/12 second vaccine of Tsenkovsky in doses of 100 and 1000 spores.

On the fifth day after infection in laboratory animals in 62.5-75.0% of cases was identified gender is positive skin allergic reaction with intensity index from 18,8 to 23.4% for drug, obtained by the proposed method and allergen isolated from biomass, dried with acetone.

10. As a result of experiments with the use of closely related microorganisms (Bacillus cereus 720, B. megaterium Bacillus 3 and 5, Bacillus subtilis 7 and 447) proven high specificity anthrax allergen obtained by the proposed method. In all cases, the production of skin-Allergy testing positive reaction was observed (table 3).

11. When studying the persistence of liquid and dry forms of anthrax allergen obtained by the proposed method was proven stability of the basic properties of the drug for three years at a storage temperature of 2 to 10°C and one month before it is used at a temperature of from 25 to 30°C. the Activity of the allergen was freshly prepared (table 4).

12. Upon receipt of the anthrax allergen developed method was used existing power production of the anthrax vaccine, which has helped to significantly reduce the financial costs of its production.

According to the results of the experimental work was prepared regulatory documentation for production and quality control of anthrax allergen, in accordance with which the apparatus-technological line for the production of anthrax vaccine were developed series of drug. It was found that during one cycle duration of 14-16 days you can get not less than 70 thousand diagnostic doses of allergen.

The proposed method for obtaining diagnostic anthrax allergen enables its production in the required amount, thereby effectively solve the problem of assessing the strength of immunity and diagnosis of anthrax infection in persons to work with anthrax. The introduction of the drug into practice will prevent possible disease among vaccinated personnel who do not have enough busy protivovirusnogo immunity.

Diagnostic anthrax allergen in liquid and dry forms received from vegetative cells of the vaccine strain of Bacillus anthracis STI-1 without pre-drying with acetone, the main indicators of quality is not inferior to previously patented drug /1/.

SOURCES of INFORMATION

1. Vasiliev N.T., Loginov M. R., Vasilyev, p. g, Frolov, V.I., sapina L.V., Zabokrickiy A.N., Garden CENTRE, Garden E.A., Stolyarov V. M., A. A. Ilyin's cartographic establishment, Fofanov P.E. Patent for invention "Dry anthrax diagnostic allergen, No. 2314348 from 10.01.2008,

2. Shlyakhov E.N., Schwartz S.A. USSR Author's certificate No. 219753, class. And 39-35, 1968

3. Industrial regulation No. 08461522-04-07. The anthrax vaccine live lyophilisate for preparation of suspension for subcutaneous and cutaneous scarification application. Registration number Gikim. Lautareasca PR No. 1898-07.

4. Harmful substances in industry. Organic substances. A Handbook for chemists, engineers and doctors. The "Chemistry"publishing house, Leningrad branch, 1971.

5. Shlyakhov E.N. Anthrax allergen, anthracen (design principles, production methods, experimental study, applied in epidemiological practice). Abstract of Diss. Prof. M., 1965.

1. The method of obtaining diagnostic anthrax allergen in liquid and dry forms, providing for the preparation of seed culture production strain Bacillus anthracis STI-1, the cooking liquid nutrient medium on the basis of means of hydrochloric acid hydrolyzed fish meal, obtaining bacterial mass vegetative cells by the method of deep cultivation, laundering on the separator and obtaining a concentrated suspension of vegetative cells, removing the allergenic protein fractions by the method of alkaline hydrolysis, fractionation of the resulting hydrolyzate, the allocation of a protein allergenic fraction by fractionation of the supernatant liquid solution of acetic acid, dissolving the precipitate obtained in sterile 0.9 percent aqueous sodium chloride solution followed by dialysis liquid substance allergen in sleeves viscose tube and lyophilization, to obtain a liquid form, a diagnostic tool for the CSOs anthrax lyophilized allergen protein allergenic fraction dissolved in borate buffer solution, autoclave, filtered through a membrane "Vladipor", sterile solution is poured into ampoules and sealed, and to obtain the dry form, sterile solution in vials freeze-dried.

2. The method according to claim 1, characterized in that the cultivation of vegetative cells of strain Bacillus anthracis STI-1 is performed with the use of liquid nutrient medium on the basis of 1% means of hydrochloric acid hydrolyzed fish meal and corn extract containing total nitrogen from 1.2 to 1.6 g/DM³, amino nitrogen from 0.3 to 0.5 g/DM³, reducing substances from 3.9 to 4.1 g/DM³, chlorides from 1.6 to 3.0 g/DM³and having a pH of from 7.5 to 7.9.

3. The method according to claim 1, characterized in that the preparation was washed concentrated suspension of vegetative cells carry on the separator VDR-3MB dual shaded cells purified water when serving in mesterolone space drum separator native vegetative culture at speeds from 30 to 40 DM³/PM

4. The method according to claim 1, characterized in that the selection of allergenic protein fraction is carried out directly from the washed concentrated suspension of vegetative cells vaccine strain of Bacillus anthracis STI-1 using the alkaline hydrolysis of a 1%solution of potassium hydroxide at the rate of 100 cm³alkali (12±1) g cells during 25-29 hours at a temperature of from 20 to 24°C With periodic p is remesiana 3-4 hours for 5 min and subsequent fractionation of the supernatant liquid after adding a 50%aqueous solution of acetic acid at the rate of 1 volume of acid on the 4th volume of the hydrolysate.