RU2416429C2 - Method for producing antigen preparation of l-brucellas - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and veterinary science, namely to microbiology and immunology. The method involves cultivation of Brucella abortus I-206 L-strain on a dense nutrient medium for L-brucellas at 37°C for 3-5 days. Thereafter, a microbial mass is washed of with buffered 0.9% normal saline of pH 7.2±0.2 and inactivated by adding 2.5% formalin. A bacterial suspension is kept for one day at 37°C with specific sterility being controlled. The bacterial suspension is reduced to concentration 4.5·10¹⁰-5·10 m.c. in ml with buffered 0.9% normal saline (pH 7.2±0.2) and thermally treated on a water bath at 100°C for 45-50 minutes. The suspension is centrifuged at 7000 rpm for 50-60 minutes; the supernatant is separated and frozen-dried. The method allows producing the preparation exhibiting high specificity and activity, available for preparing based immunobiological preparations and a test-system for human and animal blood serum examination for L-brucella antibodies.

EFFECT: method is technological, accessible, does not require using expensive devices and equipment.

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Description

 The invention relates to the field of medicine and veterinary medicine, namely to microbiology and immunology, and can be used in the production of medical and veterinary immunobiological preparations.

The persistence of L-forms of brucella in humans and animals, followed by reversion to a typical form, may be one of the causes of unpredictable epizootological and epidemiological complications. When identifying non-manifest foci of infection and deciphering atypically occurring forms of the disease in people, the most informative methods of laboratory diagnosis of brucellosis are crucial. To date, test systems for the diagnosis of brucellosis due to L-forms of the pathogen have not been produced. Therefore, the development of such test systems using specific drugs from L-forms of brucella is relevant.

The aim of the invention is to develop a method for producing an antigenic preparation from brucella in L-form, suitable for creating on its basis immunobiological preparations for the determination of antibodies against brucella in L-form in the blood serum of humans and animals.

This goal is achieved in that cells of the Brucella abortus I-206 strain are cultured in L-form on a solid nutrient medium for culturing brucella in L-form at 37 ° C for 3-5 days. The microbial suspension is washed off with a buffered 0.9% sodium chloride solution (pH 7.2 ± 0.2) and inactivated by the addition of 2.5% (vol./about.) Formalin, after which it is kept at 37 ° C for a day. After controlling for specific sterility, a suspension of brucella is prepared according to the standard turbidity of bacterial suspensions of 10 units. GISK in a concentration of 4.5 · 10 ¹⁰ -5 · 10 ¹⁰ mk in ml Next, the suspension is subjected to heat treatment in a water bath at 100 ° C for 45-50 minutes. After cooling, the suspension is centrifuged at 7000 rpm for 50-60 minutes. The supernatant is separated, poured into glass ampoules of 1 ml and freeze-dried.

In the available literature, the authors have not found ways to obtain an antigenic drug from brucella in L-form. The proposed technical solution is based on the fact that to obtain the drug, a strain of the brucellosis microbe Brucella abortus I-206 in L-form is used. This strain is cultivated on a solid nutrient medium for culturing brucella in L-form at 37 ° C for 3-5 days. The microbial suspension is washed off with a solid nutrient medium with a buffered 0.9% sodium chloride solution (pH 7.2 ± 0.2) and inactivated by the addition of 2.5% (vol./about.) Formalin, after which it is kept at 37 ° C for 24 hours. After controlling for specific sterility, a suspension of brucella is prepared according to the standard turbidity of bacterial suspensions of 10 units. GISK in a concentration of 4.5 · 10 ¹⁰ -5 · 10 ¹⁰ mk in ml Next, the suspension is subjected to heat treatment in a water bath at 100 ° C for 45-50 minutes. After cooling, the suspension is centrifuged at 7000 rpm for 50-60 minutes. The supernatant is separated, poured into glass ampoules of 1 ml, freeze-dried and sealed.

Thus, the proposed technical solution meets the criteria of the invention of "novelty."

The analysis of patent and specialized literature showed that the proposed method differs from technical solutions in this and related fields. The proposed modes allow us to achieve the goal - to obtain a highly specific and active drug from brucella in the L-form, suitable for creating immunobiological preparations on its basis for the determination of antibodies against brucella in the L-form in the blood serum of people and animals suffering from brucellosis.

This method of obtaining a drug from brucella in L-form can be used in the production of immunobiological preparations used in medical and veterinary practice.

Thus, the proposed method for producing an antigenic preparation meets the criteria of "inventive step" and "industrial applicability".

The method is as follows: cultivate cells of the strain Brucella abortus I-206 in L-form on a solid nutrient medium for culturing brucella in L-form at 37 ° C for 3-5 days. The strain Brucella abortus I-206 in L-form is in the collection of the Museum of Living Cultures of the Irkutsk Research Anti-Plague Institute of Siberia and the Far East of Rospotrebnadzor.

The nutrient medium for the cultivation of brucella in L-form contains the following components (wt.%): Agar-agar 1,2, sodium chloride 0.1, magnesium sulfate 0.05, sucrose 10, glycerol 1.0, normal horse serum 10, bicillin - 3 (5-9) · 10 ³ U / ml, hepatic infusion - the rest.

The microbial suspension is washed off with a 0.9% solution of sodium chloride, buffered with phosphate-acid salts of sodium and potassium (disubstituted phosphate-acid sodium and monosubstituted phosphate-acid potassium), pH 7.2 ± 0.2 and inactivated by the addition of 2.5% (about ./ob.) formalin, after which it is kept at 37 ° C for a day. After controlling for specific sterility, a suspension of brucellas is prepared using 0.9% buffered with phosphate-acid sodium and potassium salts (disubstituted phosphate-acid sodium and monosubstituted phosphate-acid potassium) sodium chloride solution, pH 7.2 ± 0.2, according to the turbidity standard bacterial suspensions of 10 units. GISK in a concentration of 4.5 · 10 ¹⁰ -5 · 10 ¹⁰ mk in ml Next, the suspension is subjected to heat treatment in a water bath at 100 ° C for 45-50 minutes. After cooling, the suspension is centrifuged at 7000 rpm for 50-60 minutes. The supernatant is separated, poured into glass ampoules of 1 ml, freeze-dried, after which the ampoules are sealed.

In 1 ml of the preparation obtained by the proposed method, the protein content was 603.2-648.6 μg, carbohydrates - 785-844.1 μg, total nucleic acids 9.5-10.2 μg. SDS-electrophoresis in the preparation, reprecipitated with ethanol, showed the presence of 11 components stained with protein dye Kumasi bright blue R-250, of which two major polypeptides with mol. masses of 62.8-67.5 and 69.3-74.5 kDa. To create a suspension erythrocyte test system for RPHA, the drug in the amount of 7500 (150.8 μg of protein) and 10000 μg (162.2 μg of protein) was conjugated with 1 ml of 5% sheep erythrocytes.

The resulting preparation was tested for specificity and activity in the reactions of ring precipitation (PK), gel immunodiffusion (RID) and passive hemagglutination reaction (RPHA).

The activity of the drug in the reaction of ring precipitation with hyperimmune rabbit serum against brucella in the L-form was determined by the formation of a specific ring at the border of the drug and serum (positive reaction) when the drug was diluted to 1: 160. An undiluted preparation reacted positively with a specific anti-brucella antiserum in L-form diluted to 1:10. In the RID gel, the drug, with a load of 2-3 mg per well, gave a positive reaction with hyperimmune rabbit serum against brucella in L-form to a dilution of 1: 8-1: 16, respectively. Homologous serum RPHA was positive before a 1: 12800 dilution.

The specificity of the drug was determined with hyperimmune serums against brucella and some bacteria that have common antigenic determinants and give cross-reactions: brucellosis against the original strain in S-form, cholera diagnostic “O”, and tularemia diagnostic. There is no specific interaction of the drug in RK and RID with the indicated sera, in RPGA there is a weak interaction with polyvalent brucellosis serum (S) before a dilution of 1: 200, which indicates its high specificity.

Obtained by the proposed method, the drug was used to study the blood serum of patients with chronic brucellosis in humans. In 4.7% of cases, positive results were obtained in the Republic of Kazakhstan, positive reactions were noted in RPHA up to titers of 1: 800-1: 1600, indicating the presence of antibodies against brucella in their L-form in their blood serum.

Example 1

Cells of the Brucella abortus I-206 strain in L-form were grown on solid nutrient medium for culturing brucella in L-form at 37 ° C for 3 days, while the nutrient medium contained bicillin-3 in an amount of 5 · 10 ³ U / ml. The microbial suspension was washed off with a buffered 0.9% sodium chloride solution pH 7.0 and inactivated by the addition of 2.5% (vol./about.) Formalin, and then kept at 37 ° C for a day. After controlling for specific sterility, a brucella suspension was prepared using a buffered 0.9% sodium chloride solution pH 7.0, according to the standard turbidity of bacterial suspensions of 10 units. GISK in a concentration of 4.5-10 ¹⁰ mk / ml. Next, the bacterial suspension was subjected to heat treatment in a water bath at 100 ° C for 45 minutes. After cooling, the suspension was centrifuged at 7000 rpm for 50 minutes. The supernatant was separated, poured into 1 ml glass ampoules and freeze-dried.

In 1 ml of the preparation obtained, the protein content was 603.2 μg, carbohydrates - 785 μg, and total nucleic acids 9.5 μg. SDS-electrophoresis in the preparation, reprecipitated with ethanol, showed the presence of 11 components stained with protein dye Coomassie bright blue R-250, of which two major polypeptides with mol. masses of 62.8 and 69.3 kDa. To create a suspension erythrocyte test system for RPHA, the drug in the amount of 7500 μg (150.8 μg of protein) was conjugated with 1 ml of 5% sheep erythrocytes.

The resulting preparation was tested for specificity and activity in the reactions of ring precipitation (PK), gel immunodiffusion (RID) and passive hemagglutination reaction (RPHA).

A positive reaction of the drug in RK with hyperimmune rabbit serum against brucella in L-form was recorded when the drug was diluted to 1: 160. An undiluted preparation reacted positively with a specific anti-brucella antiserum in L-form diluted to 1:10. In the RID gel, the drug, with a 2 mg load per well, gave a positive reaction with hyperimmune rabbit serum against brucella in L-form before dilution of 1: 8. Homologous serum RPHA was positive before a 1: 12800 dilution.

The specificity of the drug was determined with hyperimmune serums against brucella and some bacteria that have common antigenic determinants and give cross-reactions: brucellosis against the original strain in S-form, cholera diagnostic “O”, and tularemia diagnostic. The results obtained showed the absence of a specific interaction of the drug in RK and RID with the indicated sera; in RPGA, a weak interaction with polyvalent brucellosis serum (S) was noted before dilution of 1: 200, which indicates its high specificity.

Example 2

Cells of the Brucella abortus I-206 strain in L-form were grown on solid nutrient medium for cultivation of brucella in L-form at 37 ° C for 5 days, while the nutrient medium contained bicillin-3 in the amount of 9 · 10 ³ U / ml. The microbial suspension was washed off with a buffered 0.9% solution of sodium chloride pH 7.4 and inactivated by the addition of 2.5% (vol./about.) Formalin, and then kept at 37 ° C for a day. After controlling for specific sterility, a brucella suspension was prepared using a buffered 0.9% sodium chloride solution, pH 7.4, according to the standard turbidity of bacterial suspensions of 10 units. GISK in a concentration of 5 · 10 ¹⁰ mk./ml Next, the bacterial suspension was subjected to heat treatment in a water bath at 100 ° C for 50 minutes. After cooling, the suspension was centrifuged at 7000 rpm for 60 minutes. The supernatant was separated, poured into 1 ml glass ampoules and freeze-dried.

In 1 ml of the obtained preparation, the protein content was 648.6 μg, carbohydrates - 844.1 μg, total nucleic acids 10.2 μg. SDS-electrophoresis in the preparation, reprecipitated with ethanol, showed the presence of 11 components stained with protein dye Coomassie bright blue R-250, of which two major polypeptides with mol. masses of 67.5 and 74.5 kDa. To create a suspension erythrocyte test system for RPHA, the drug in an amount of 10,000 μg (162.2 μg of protein) was conjugated with 1 ml of 5% ram erythrocytes.

The resulting preparation was tested for specificity and activity in the reactions of ring precipitation (PK), gel immunodiffusion (RID) and passive hemagglutination reaction (RPHA)

A positive reaction of the drug in RK with hyperimmune rabbit serum against brucella in the L-form was recorded when the drug was diluted to 1: 160. An undiluted preparation reacted positively with a specific anti-brucella antiserum in L-form diluted to 1:10. In the RID gel, the drug, with a load of 3 mg per well, gave a positive reaction with hyperimmune rabbit serum against brucella in L-form to a 1:16 dilution. Homologous serum RPHA was positive before a 1: 12800 dilution.

The specificity of the drug was determined with hyperimmune serums against brucella and some bacteria that have common antigenic determinants and give cross-reactions: brucellosis against the original strain in S-form, cholera diagnostic “O”, and tularemia diagnostic. The results obtained showed the absence of a specific interaction of the drug in RK and RID with the indicated sera; RPGA showed a weak interaction with polyvalent brucellosis serum (S) before a dilution of 1: 200, which indicates a high specificity of the obtained preparation.

Thus, the proposed method for producing an antigenic drug is technologically advanced in production, does not require the use of expensive instruments and equipment and allows you to get a drug that has high specificity, activity, which allows you to create immunobiological preparations and test systems for determining antibodies in human serum and animals against brucella in L-shape.

Claims (2)

1. A method of obtaining an antigenic preparation from brucella in L-form, comprising cultivating a strain of Brucella abortus I-206 in L-form on a solid nutrient medium for brucella in L-form at 37 ° C for 3-5 days, washing off the microbial mass with a buffered 0.9% sodium chloride solution with a pH of 7.2 ± 0.2, inactivation by adding 2.5% formalin, keeping the bacterial suspension at 37 ° C for a day, monitoring specific sterility, bringing the bacterial suspension to a concentration of 4.5 · 10 ¹⁰ -5 · 10 ¹⁰ m.k. in ml with a buffered 0.9% sodium chloride solution (pH 7.2 ± 0.2), heat treatment of the suspension in a water bath at 100 ° C for 45-50 minutes, centrifugation of the suspension at 7000 rpm for 50 -60 min, separation and lyophilization of the supernatant. 2. The method according to claim 1, characterized in that after separation, the supernatant is poured into 1 ml.