RU2361610C1 - Way of reception of brucellous antigen from strain brucella abortus 19 for manufacturing of uniform brucellous antigen for aa (agglutination assay), cfr (complement fixation reaction) and lcfr (long-term complement fixation reaction), brucellous antigen for rose bengal assay (rba) and brucellous antigen for ring test (rt) with milk, way of manufacturing of brucellous diagnostic serum and diagnostic sets - Google Patents

Abstract

FIELD: medicine, veterinary science.

SUBSTANCE: group of inventions concerns veterinary science and biotechnology area. The way of reception of a brucellous antigen from the Brucella abortus 19 strain for manufacturing of a uniform brucellous antigen for AA, CFR and LCFR includes reception of an inoculum and propagation of brucellas in deep conditions on a liquid nutrient medium with regulation of level of partial pressure of oxygen dissolved in a culture liquid throughout all propagation process, separation and concentration of evolved bacteriemic cells using ultrafiltration and their inactivation using warming. Thus brucellas propagation in deep conditions on a liquid nutrient medium with regulation of level of partial pressure of oxygen dissolved in a culture liquid throughout all propagation process is performed in a following regimen: temperature 37±0.5°C, pH 6.9-7.2 aeration from 1 hour till 3 hour - 20-25 pO₂, from 3 hour till 6 hour - 25-40 pO₂, from 6 hour till 15 hour - 40-45 pO₂ and from 15 hour till 16-18 hour - 30-35 pO₂. Separation and concentration of the evolved bacteriemic cells by ultrafiltration is carried out with use of membranous cartridges. The concentrate of an antigen received by given way is used also for reception of an antigen for Rose Bengal Assay (RBA), for reception of a brucellous antigen for ring test (RT) with milk and reception of diagnostic brucellous serum which, together with blood serum of healthy livestock, make a part of sets for brucellosis diagnostics. The group of inventions allows to raise efficiency of process of brucellosis diagnostics and to improve control at serologic examination of animals.

EFFECT: rising of efficiency of process of brucellosis diagnostics and improvement of control at serologic examination of animals

22 cl, 11 ex

Description

The group of inventions relates to the fields of veterinary medicine and biotechnology.

A known method of producing brucellosis antigen from strain Brucella abortus 19 for the manufacture of a single brucellosis antigen for RA, PCK and RDSK, including the cultivation of the causative agent of brucellosis in a solid nutrient medium in quarters (see, for example, Kasyanov AN and others. Obtaining a single brucellosis antigen for PA, PCK and RDSK, Veterinary, 1973, No. 10, S. 50-52).

The method requires the use of an expensive, difficult to manufacture dense nutrient medium, a large number of glassware, when it is used, there is the possibility of personnel contact with a living culture of brucella.

A known method of producing brucellosis antigen from strain Brucella abortus 19 for the manufacture of a single brucellosis antigen for RA, PCK and RDSK, including obtaining seed in solid nutrient medium, deep cultivation of strain Br.abortus 19 in a liquid nutrient medium, concentration and purification in an ultrafiltration unit and inactivation of the antigen in the bioreactor at a temperature of 80 ° C for an hour (see, for example, RU 2085212 C1, 07.27.1997). This method is time consuming, because involves the preparation of seed in a dense nutrient medium and the concentration of the bacterial mass in two doses.

A known method of producing an antigen for serological diagnosis of brucellosis, according to which the vaccine strain Brucella abortus 19 and brucellosis bacteriophage are used for the production of antigen (see, for example, SU 1713592 A, 23.02.1992). The antigen obtained by this method is active, has a high sensitivity, however, the method for its preparation is complex, requires a large consumption of bacterial material, the presence of a specific brucellosis bacteriophage, the operation of centrifuging the suspension is dangerous for staff.

The closest is a method for producing brucellosis antigen from Brucella abortus 19 strain for the manufacture of a single brucellosis antigen for PA, PCK and RDSK, which includes obtaining seed and culturing the causative agent of brucellosis in a deep nutrient medium with regulation of the level of partial pressure of oxygen dissolved in the culture fluid on throughout the cultivation process, the separation and concentration of grown bacterial cells by ultrafiltration on fibers and their inactivation on Revani (see., e.g., RU 2163141 C1, 20.02.2001). However, this method is long, because the stage of cultivation of the causative agent of brucellosis is 19-21 hours, the stage of concentration of bacterial cells is 5-7 hours, and during ultrafiltration, fiber breakage is often observed, which leads to the loss of bacterial mass and its contamination by extraneous microflora.

A known method of producing brucellosis antigen for rose bengal test (RBP), including staining the antigen of brucellosis Bengal pink (see, for example, Veterinary preparations. Reference book. M .: Kolos. P. 185-187).

A known method of producing brucellosis antigen for a ring reaction (CR) with milk, comprising staining the brucellosis antigen with hematoxylin in blue or tetrazolium in red-cherry color (see, for example, Veterinary preparations. Reference book. M .: Kolos. Pages 183-185) .

The objective of the group of inventions in terms of a method for producing a brucellosis antigen from a strain of Brucella abortus 19 for the manufacture of a single brucellosis antigen for RA, RSK and RDSK, a brucellosis antigen for RBP, a brucellosis antigen for KR with milk is to unify and simplify the technology and reduce the time for producing the antigen for preparations a single brucellosis antigen for RA, RSK and RDSK, a brucellosis antigen for RBP, a brucellosis antigen for CR with milk, increasing its quality and increasing the culture of its production.

The problem in terms of the method of producing brucellosis antigen from Brucella abortus 19 strain for the manufacture of a single brucellosis antigen for RA, RSK and RDSK is solved by the fact that in the method for producing brucellosis antigen from the Brucella abortus 19 strain for the manufacture of a single brucellosis antigen for RA, RSK and including the preparation of seed and cultivation of the causative agent of brucellosis in deep conditions on a liquid nutrient medium with the regulation of the level of partial pressure of oxygen dissolved in the culture fluid over During the whole cultivation process, separation and concentration of the grown bacterial cells by ultrafiltration and their inactivation by heating, according to the invention, the cultivation of the causative agent of brucellosis in deep conditions on a liquid nutrient medium with the regulation of the partial pressure level of oxygen dissolved in the culture fluid during the entire cultivation process is carried out in the following mode: temperature 37 ± 0.5 ° C, pH 6.9-7.2, aeration - from 1 to 3 hours - 20-25 pO ₂ , from 3 to 6 hours - 25-40 pO ₂ , from 6 to 15 hours - 40 -45 pO ₂ and from 15 to 16-18 ca - 30-35 pO _2, and the separation and concentration of bacterial cells grown ultrafiltration is carried out using membrane cassettes.

The problem is also solved by the fact that the concentration of seed is 3.5-5.0 billion cubic meters / cm ³ .

The problem is also solved by the fact that inactivation of microbial cells by heating is carried out after their cultivation in the fermenter at a more sparing antigen mode: temperature - 70 ± 1 ° C, time - 1 hour.

The problem is also solved by the fact that the cultivation of the causative agent of brucellosis in deep conditions is carried out for 16-18 hours.

The problem is also solved by the fact that in the concentration using membrane cassettes with a retention limit of 20 KD.

The problem is also solved by the fact that during concentration they use membrane cassettes with a filter holder ASF-007.

The problem is also solved by the fact that the concentration is carried out for 2-2.5 hours.

The problem is also solved by the fact that at the end of the concentration, the resulting antigen is resuspended in a sterile 1% phenolized isotonic solution to an OK of 250-300 billion m3 / cm ³ .

The task in terms of a method for producing brucellosis antigen from a strain of Brucella abortus 19 for the manufacture of brucellosis antigen for rose bengal samples (RBP) is solved by the fact that in the method for producing brucellosis antigen for RBP, including staining a Bengal pink antigen concentrate, the invention uses an antigen concentrate according to the invention obtained as described above.

The problem is also solved by the fact that Bengal pink is titrated in a water bath at a temperature of 40 ° C before staining, and the antigen is stained at the same temperature for an hour.

The problem in terms of a method for producing brucellosis antigen from a strain of Brucella abortus 19 for the manufacture of brucellosis antigen for a ring reaction (CR) with milk is solved by the fact that in a method for producing a brucellosis antigen for a ring reaction with milk, including staining the resuspended bacterial mass of the antigen with tetrazolate chloride or chloride , according to the invention use the antigen obtained as described above.

The technical result obtained by using the claimed group of inventions is an increase in the accumulation of brucella while reducing cultivation time, increasing agglutinable properties while reducing the concentration of microbial cells in the finished product, reducing the time of concentration of bacterial cells and eliminating their contamination by extraneous microflora during the concentration process, as well as eliminating contact personnel with a lively Brucella culture while concentrating on an ultrafiltration unit and schenie time Brucella antigen staining for BPO.

A known method for producing monospecific sera by immunizing rabbits intravenously with single doses of antigen from strain Br.abortus in a dose containing 10 ⁹ CFU in 1 ml Then, 5-7 days after the completion of the immunization, the animals are bled at a titer of serum agglutination of at least 1: 1280, after which the serum is absorbed by the Bakassa strain of the heterologous type of brucella, incubated at 37 ° C for 2 hours, restored by centrifugation, diluted in phenol salt solution, Packed and stored at 4 ° C (see, e.g., Corbel MJ et al., Methods for the Identification of Brucella. Ministry of Agriculture, Floweriest and Food. Research Section. Central veterinary Lab. New. Ham. Weighbridge. Surry RT, 153, 13B, 1978).

The disadvantage of this method is the low sensitivity of the resulting serum.

A known method for producing diagnostic brucellosis agglutinating serum, which consists in immunizing rabbits producing live virulent cells of B. abortus 146 and B. melitensis I-297. Chinchilla rabbits weighing 2.5-3.0 kg are immunized with a suspension of living cells in a 0.85% sodium chloride solution of a 48-hour brucella agar culture. Animals are injected subcutaneously three times at four points of the back along the spine with 0.5, 0.75 and 1.0 ml of the mixture in equal amounts of cultures with an equal volume of Freund's complete adjuvant with a weekly interval. Two weeks after the final injection of antigen, subcutaneously injected into the region of the inner upper third of the thigh surface 1.0 ml of a mixture of cultures in a 0.85% sodium chloride solution. 14 days after the end of immunization, rabbits are totally bled. Serum is inactivated by heating and canned by the addition of sodium thiolate (see, for example, RU 2005781 C1, 01/15/1994).

When using a live virulent culture, a strict anti-epidemic regime is necessary, the method is time-consuming and time-consuming.

The closest is a method of obtaining a multivalent diagnostic brucellosis serum, which cattle are immunized at the age of 2 to 5-6 years, while non-castrated producing bulls are used. Prior to the start of immunization, the animals are kept in quarantine for 1 month, after which the immunization is carried out by single injection of 10 ml of brucellosis antigen subcutaneously, and after 1 month immunization is started. Immunization is carried out in 2-4 cycles at intervals of 20-30 days. During one cycle, 5 daily injections of antigen are carried out with volumes of 10, 15, 20, 30, 40 ml. At the end of the immunization cycle on the 10th day after the last injection of antigen, a control blood sample is taken to set the agglutination reaction. In the presence of an antibody titer in blood serum of not less than 1: 3200, bloodletting is performed. To immunize an animal, antigens are used, inactivated by heating at a temperature of 60 ° C for 2 hours, followed by the addition of carbolic acid and keeping at 37 ° C for 48 hours. Antigens are suspensions of cultures of B. abortus 544, B. melitensis 16-M and B. suis 1330 (see, for example, Diagnostic brucellosis agglutinating serum. TU-316-82 from 06/01/1982, approved by the State Institution for the production of bacterial and viral drugs of the Ministry of Health of the USSR).

The disadvantages of the method are the duration, the complexity of the process, the epidemic hazard associated with the use of virulent cultures of brucella, and the high cost of the final product.

The objective of the invention in terms of a method for producing brucellosis diagnostic serum is to reduce the complexity of the manufacturing process and increase serum activity.

The problem in terms of the method for producing brucellosis diagnostic serum is solved by the fact that the method for producing brucellosis diagnostic serum, including hyperimmunization of animal producers with brucellosis antigen, followed by bloodletting, separation of serum, preservation, testing for sterility, activity and specificity, and packaging, is solved that according to the invention, hyperimmunization of animal producers is carried out by an antigen (live brucella culture) obtained by the method described mu above.

As animal producers use oxen.

Hyperimmunization of animal producers is carried out with an antigen made from a vaccine strain of B. abortus 19, subcutaneously in the middle third of the neck according to the following scheme: 1st day - 5 cm ^{3 of} antigen (75 billion cubic meters) are administered, 10th day - they inject 8 cm ^{3 of} antigen (120 billion cubic meters), day 20-21 - they inject 10 cm ^{3 of} antigen (150 billion cubic meters), test bleeding is carried out on days 27-28, and 30 -31st day - carry out industrial bloodletting.

The blood is kept at a temperature of 37-38 ° C for 2-3 hours, and then placed in a refrigerator at 2-8 ° C for 3-5 days, after which the serum is separated.

The resulting serum is preserved with a 5% phenol solution in an isotonic solution in a ratio of 1:10.

The resulting serum is preserved by adding 4% dry boric acid, and then heated in a water bath at a temperature of 54-56 ° C for 40 minutes with constant stirring, and then placed in a refrigerator at a temperature of 2-8 ° C for 10 days.

Serum must be sterile, have a titer in RA of not less than 1: 1000, and in CSC - not less than 1:20.

After filling the serum, it is lyophilized.

The technical result achieved by using the claimed method is that the cycle time of hyperimmunization of animals is significantly reduced, the method for producing serum is simplified, and its cost is significantly reduced with increasing activity. The method eliminates the possibility of infection of personnel.

The sets of components for the diagnosis of brucellosis in the prior art are unknown.

The objective of the invention in terms of sets of components for the diagnosis of animal brucellosis is to improve control during serological testing of animals for brucellosis.

The set of components for the diagnosis of animal brucellosis in PA, PCK and RDSK according to the claimed invention contains a single brucellosis antigen for RA, PCK and RDSK obtained by the method described above, diagnostic brucellosis lyophilized serum obtained by the method described above, and blood serum of healthy cattle lyophilized.

The kit of components for diagnosing animal brucellosis in a rose bengal sample (RBP) according to the claimed invention contains a brucellosis antigen for RBP obtained by the method described above, lyophilized brucellosis diagnostic serum obtained by the method described above, and blood serum of healthy cattle lyophilized.

The set of components for the diagnosis of animal brucellosis in a ring reaction (CR) with milk according to the claimed invention contains brucellosis antigen for CR obtained by the method described above, and lyophilized diagnostic brucellosis serum obtained by the method described above.

The technical result of the group of inventions in terms of sets is that the claimed set of sets allows to unify the serological diagnosis of animal brucellosis and to control the correctness of the formulation of serological reactions in all veterinary laboratories of the country.

The group of inventions is characterized by the following examples.

Example 1

To obtain seed and industrial cultivation of brucella, a sterile nutrient medium based on the Hottinger digest is used, containing, vol.%: Hottinger digest - 25, glycerin - 2, glucose - 1.5, sodium chloride - 0.5, native yeast autolysate - 3 , 4, water - up to 100. To obtain seed, a pure culture of the strain Brucella abortus 19 is seeded in microfermenter with this nutrient medium. The seed concentration is 3.5 billion m.cl./cm ³ . Cultivate 24 hours before the accumulation of 35 billion m.cl./cm ³ .

Production cultivation is carried out by the in-depth method in a bioreactor in a nutrient medium of the same composition. During cultivation, the pH is maintained within 6.9, and the temperature within 36.5 ° C. The partial pressure level of oxygen dissolved in the culture fluid is: from 1 to 3 hours - 20 pO ₂ , from 3 to 6 hours - 25 pO ₂ , from 6 to 15 hours - 40 pO ₂ and from 15 to 16 hours - 30 pO ₂ . The cultivation time is 16 hours, the accumulation of cells is 70 billion m.cl./cm ³ . At the end of cultivation, the bacterial suspension is concentrated by ultrafiltration using membrane cassettes with a retention limit of 20 KD. The concentration time is 2.5 hours. The concentrate in the bioreactor is resuspended with a sterile 1% phenolized isotonic solution up to 250 billion cubic meters / cm ³ and inactivated by heating in the following mode: temperature - 70 ± 1 ° C, time - 1 hour.

The inactivated bacterial suspension is poured into sterile bottles and stored in the refrigerator.

The resulting bacterial suspension concentrate is used for the manufacture of a single brucellosis antigen for PA, PCK and RDSK, as well as for the production of brucellosis antigen for a rose bengal sample (RBP) and for the production of brucellosis antigen for a ring reaction (CR) with milk.

Example 2

The antigen is made analogously to example 1, but to obtain seed and industrial cultivation of brucella, a sterile nutrient medium based on the Hottinger digest is used, containing, vol.%: Hottinger digest - 20, glycerin - 1, glucose - 1.0, sodium chloride - 0, 5, native yeast autolysate - 3.0, water - up to 100. To obtain seed, a pure culture of strain Brucella abortus 19 is seeded in microfermenter with this nutrient medium. The seed concentration is 5.0 billion m.cl./cm ³ . Cultivate 26 hours before the accumulation of 45 billion m.cl./cm ³ .

Production cultivation is carried out by the in-depth method in a bioreactor in a nutrient medium of the same composition. During cultivation, the pH is maintained within 7.2, and the temperature within 37.5 ° C. The partial pressure level of oxygen dissolved in the culture fluid is: from 1 to 3 hours - 25 pO ₂ , from 3 to 6 hours - 40 pO ₂ , from 6 to 15 hours - 45 pO ₂ and from 15 to 18 hours - 35 pO ₂ . The cultivation time is 18 hours, the accumulation of cells is 75 billion m.k./cm ³ . At the end of the cultivation, the bacterial suspension is concentrated by ultrafiltration using membrane cassettes with an ASF-007 filter holder. The concentration time is 2.0 hours. The concentrate in the bioreactor is resuspended with a sterile 1% phenolized isotonic solution up to 300 billion cubic meters / cm ³ , inactivated by heating in the following mode: temperature - 70 ± 1 ° C, time - 1 hour.

The inactivated bacterial suspension is poured into sterile bottles and stored in the refrigerator.

The resulting bacterial suspension concentrate is used for the manufacture of a single brucellosis antigen for PA, PCK and RDSK, as well as for the production of brucellosis antigen for a rose bengal sample (RBP) and for the production of brucellosis antigen for a ring reaction (CR) with milk.

Example 3

The activity and specificity of the antigen obtained in Examples 1 and 2 was established by titration in RA with a standard serum sample against Brucella abortus. The proposed antigen containing a smaller number of cells (17.3-17.5 billion mcl / cm ³ ) gives a reaction of 2 cross intensities with a dilution of serum of 1: 500 (final dilution of 1: 1000), which indicates compliance with the international standard.

Example 4

Obtaining brucellosis antigen for rose bengal test (RBP).

To obtain a brucellosis antigen for a rose bengal sample (RBP), an antigen concentrate made according to Example 1 or Example 2 is placed in a biomixer. Bengal pink is titrated in a water bath at a temperature of 40 ° C. Antigen staining is carried out at the same temperature. The result is a fast, within an hour, full bright and uniform staining of the microbial cell.

Example 5

The activity and specificity of the antigen obtained in Example 4 is established by titration in RBP with a standard serum sample against Brucella abortus. The proposed antigen containing a smaller number of cells (95 billion cubic meters / cm ³ ) gives a reaction of 4 cross intensities with a 1:10 dilution serum containing 100 ME, 1-2 crosses with a 1:20 dilution serum ME) and a negative reaction with serum at a dilution of 1:35 (28.5 ME), which indicates its standard.

Example 6

Obtaining brucellosis antigen for a ring reaction (CR) with milk.

To obtain a brucellosis antigen for a ring reaction (CR) with milk, the resuspended bacterial mass of the antigen made in accordance with Example 1 or Example 2 is stained with hematoxylin blue or tetrazolium in red-cherry color. The activity and specificity of the antigen obtained in this example is established by titration with 10 samples of fresh fresh or chilled cow's milk containing 2 cm ^{3 of} 40, 20 and 10 ME antibodies. The proposed antigen contains a smaller number of cells (95-100 billion m.cl./cm ³ ), which leads to its higher activity compared to the known diagnosticum.

Example 7

Obtaining brucellosis diagnostic serum.

To obtain brucellosis diagnostic serum, hyperimmunization of animal producers is carried out with the antigen (live brucella culture) obtained according to example 1 or example 2. Oxen are used as producers. Hyperimmunization of animals is carried out subcutaneously in the region of the middle third of the neck according to the following scheme: 1st day - 5 cm ^{3 of} antigen (75 billion m3) are administered, 10th day -

8 cm ³ (120 billion cubic meters), the 20th day - 10 cm ³ (150 billion cubic meters). Blood sampling is carried out on day 27, and on the 30th day, if the level of antibodies in RA is sufficient, bloodletting is performed. Blood is kept at a temperature of 37 ° C for 3 hours, and then placed in a refrigerator at 2 ° C for 3 days, after which the serum is separated.

The resulting serum canned 5% phenol solution in saline in a ratio of 1:10.

The serum is sterile, has a titer in RA of 1: 1000, and in CSC - not less than 1:20.

Ready whey is packaged and lyophilized.

Example 8

Obtaining brucellosis diagnostic serum is carried out analogously to example 7, but oxen hyperimmunization is carried out subcutaneously in the middle third of the neck according to the following scheme: 1st day - 5 cm ^{3 of} antigen are injected (75 billion cubic meters), 10th day - 8 cm ³ antigen (120 billion cubic meters), the 21st day - inject 10 cm ³ antigen (150 billion cubic meters). On day 28, test bloodletting is performed, and on day 31, production bloodletting is performed. The blood is kept at a temperature of 38 ° C for 2 hours, and then placed in a refrigerator at 8 ° C for 5 days, after which the serum is separated. The resulting serum is preserved by adding 4% dry boric acid, and then heated in a water bath at a temperature of 50 ° C for 40 minutes with constant stirring, and then placed in a refrigerator at a temperature of 5 ± 3 ° C for 10 days.

The serum is sterile, has a titer in RA of 1: 1000, and in CSC - not less than 1:20.

Ready whey is packaged and lyophilized.

Example 9

A set of components for the diagnosis of animal brucellosis in PA, PCK and RDSK.

The kit includes the following components:

- single brucellosis antigen for RA, PCK, RDSK, obtained according to examples 1-2,

- brucellosis diagnostic lyophilized serum obtained according to examples 7-8,

- negative serum, which is a lyophilized blood serum of healthy cattle.

The single brucellosis antigen reveals specific antibodies in the indicated serological reactions in the blood serum of animals infected with brucellosis or immunized with agglutinogenic anti-brucellosis vaccines.

Diagnostic brucellosis serum contains specific brucellosis antibodies that give positive serological reactions with brucellosis antigen.

Negative serum, which is the blood serum of healthy cattle, does not contain specific brucellosis antibodies and does not produce serological reactions with brucellosis antigen.

Both serums are control, confirming the specificity of the diagnostic reaction and the correctness of its formulation technique.

Example 10

A set of components for the diagnosis of animal brucellosis in RBP (rose-bengal test).

The kit includes the following components:

- brucellosis antigen for RBP obtained in examples 4-5;

- brucellosis diagnostic lyophilized serum obtained according to examples 7-8;

- negative serum, which is a lyophilized blood serum of healthy cattle.

The set of components is used for express serological rapid diagnosis of brucellosis in animals unimmunized with brucellosis vaccines by the method of plate reaction of agglutination with blood serum.

Brucellosis antigen stained with Bengal pink reveals specific antibodies in the serum of animals with brucellosis in the plate reaction of agglutination.

Diagnostic brucellosis serum contains specific brucellosis antibodies that give positive serological reactions with brucellosis antigen.

Negative serum, which is the blood serum of healthy cattle, does not contain specific brucellosis antibodies and does not produce serological reactions with brucellosis antigen.

Both serums are control, confirming the specificity of the plate reaction of agglutination and the correctness of its formulation technique.

Example 11

A set of components for the diagnosis of animal brucellosis in a ring reaction (CR) with milk.

A set of components is used to study milk in a ring reaction in order to determine the welfare of herds (farms) for brucellosis in cattle (buffalo) and for rapid diagnosis of brucellosis in the sale of milk in the markets.

The kit consists of the following components:

- brucellosis antigen for CR obtained in example 6.

- brucellosis diagnostic lyophilized serum obtained according to examples 7-8.

Brucellosis antigen for CR with milk reveals specific antibodies contained in the milk of brucellosis-infected animals by the ring reaction method.

Diagnostic brucellosis serum contains specific brucellosis antibodies, which give a positive ring reaction with brucellosis antigen.

This set of kit allows you to control the specificity of the express method and the correct formulation of the reaction.

As follows from the above examples, the technical result achieved in the implementation of the claimed group of inventions is to increase the accumulation of brucella while reducing cultivation time, increasing agglutinable properties while reducing the concentration of bacterial cells in the finished antigen due to the proposed regime of deep cultivation, reducing the time of concentration of bacterial cells from 5-7 hours to 2-2.5 hours and the elimination of their contamination by extraneous microflora in the process of concentration by replacing hollow fibers with membrane cassettes and eliminating fiber breakage during filtration, as well as eliminating personnel contact with a live brucella culture during concentration on an ultrafiltration unit by inactivating them after cultivation in the fermenter.

The proposed method for staining brucellosis antigen for RBP allows to reduce the process time from four days to two hours, and the proposed technology for hyperimmunization of producers allows to increase the activity of brucellosis diagnostic serum in RA up to 1000 ME antibodies per 1 ml. The use of lyophilized form of blood serum in kits allows preserving their activity twice as long, and the introduction of antigen, positive and negative serum into kits can improve control during serological examination of animals for brucellosis and generally unify the serological diagnosis of brucellosis.

Thus, the tasks are solved.

Claims (22)

1. A method of obtaining a brucellosis antigen from a strain of Brucella abortus 19 for the manufacture of a single brucellosis antigen for RA, CSC and RDSK, comprising obtaining seed and culturing brucella in deep conditions on a liquid nutrient medium with regulation of the partial pressure level of oxygen dissolved in the culture fluid throughout cultivation process, separation and concentration of the grown bacterial cells by ultrafiltration and their inactivation by heating, characterized in that the cultivation rutsell in deep conditions on a liquid nutrient medium with regulation of the partial pressure level of oxygen dissolved in the culture fluid during the entire cultivation process is carried out in the following mode: temperature 37 ± 0.5 ° C, pH 6.9-7.2, aeration from 1 to 3 h - 20-25 pO ₂ , from 3 to 6 h - 25-40 pO ₂ , from 6 to 15 h - 40-45 pO ₂ and from 15 to 16-18 h - 30-35 pO ₂ , and separation and concentration of the grown bacterial cells by ultrafiltration is carried out using membrane cassettes. 2. The method of producing brucellosis antigen according to claim 1, characterized in that the seed concentration is 3.5-5.0 billion m.cl./cm ³ . 3. The method of producing brucellosis antigen according to claim 1, characterized in that the inactivation of microbial cells by heating is carried out after they are cultured in a fermenter in the following mode: temperature 70 ± 1 ° C, time 1 hour 4. The method of producing brucellosis antigen according to claim 1, characterized in that the cultivation of brucella in deep conditions is carried out for 16-18 hours 5. The method of producing brucellosis antigen according to claim 1, characterized in that when concentrating the bacterial mass, membrane cassettes with a retention limit of 20 KD are used. 6. The method of producing brucellosis antigen according to claim 5, characterized in that when concentrating the bacterial mass, membrane cartridges with a filter holder ASF-007 are used. 7. The method of producing brucellosis antigen according to claim 1, characterized in that the concentration is carried out for 2-2.5 hours 8. The method for producing the brucellosis antigen according to claim 1, characterized in that upon completion of the concentration, the resulting antigen is resuspended in a sterile 1% phenolized isotonic solution to an OK of 250-300 bcm / cm ³ . 9. A method of obtaining a brucellosis antigen from a strain of Brucella abortus 19 for the manufacture of brucellosis antigen for a rose bengal sample (RBP), comprising staining a pink Bengal antigen concentrate, characterized in that the antigen concentrate obtained according to any one of claims 1 to 7 is used. 10. The method of producing brucellosis antigen according to claim 9, characterized in that the Bengal pink is titrated in a water bath at a temperature of 40 ° C before staining, and the antigen is stained at the same temperature for one hour. 11. A method of obtaining a brucellosis antigen from a strain of Brucella abortus 19 for the manufacture of brucellosis antigen for a ring reaction (CR) with milk, comprising staining the resuspended bacterial mass of the antigen with tetrazolium chloride or hematoxylin, characterized in that they use the antigen obtained according to any one of claims 1- 8. 12. A method for producing brucellosis diagnostic serum, comprising hyperimmunization of animal producers with a brucellosis antigen, followed by bloodletting, separation of serum, preservation and packaging, testing for sterility, activity and specificity, characterized in that hyperimmunization of animal producers is carried out by the antigen obtained by the method described above. 13. The method of producing brucellosis diagnostic serum according to claim 12, characterized in that oxen are used as producer animals. 14. The method for producing brucellosis diagnostic serum according to claim 12, characterized in that hyperimmunization of animal producers is carried out subcutaneously in the middle third of the neck according to the following scheme: on the 1st day, 5 cm ^{3 of} antigen (75 billion m.c.cl.) Are administered The 10th day - 8 cm ³ (120 billion cubic meters), the 20-21st day - 10 cm ³ (150 billion cubic meters), on a 27-28th day they carry out test blood sampling, and on the 30-31st day they carry out industrial bloodletting. 15. The method of obtaining brucellosis diagnostic serum according to 14, characterized in that the blood is kept at a temperature of 37-38 ° C for 2-3 hours, and then placed in a refrigerator at 2-8 ° C for 3-5 days, after which separates the serum. 16. The method for producing brucellosis diagnostic serum according to claim 15, characterized in that the obtained serum is preserved with a 5% phenol solution in an isotonic solution in a ratio of 1:10. 17. The method for producing brucellosis diagnostic serum according to claim 15, characterized in that the obtained serum is canned by adding 4% dry boric acid, and then heated in a water bath at a temperature of 50 ° C for 40 min with constant stirring, and then placed in refrigerator at a temperature of 2-8 ° C for 10 days. 18. The method for producing brucellosis diagnostic serum according to claim 12, characterized in that the sterile serum has a titer in RA of not lower than 1: 1000, and in RSK not lower than 1:20. 19. The method of producing brucellosis diagnostic serum according to claim 12, characterized in that after packaging, the serum is lyophilized. 20. Diagnostic kit for the diagnosis of animal brucellosis in RA, RSK and RDSK, characterized in that it contains a single brucellosis antigen for RA, RSK and RDSK, obtained according to any one of claims 1 to 8, diagnostic brucellosis serum lyophilized obtained according to any one of paragraphs .12-19, and healthy cattle lyophilized blood serum. 21. Diagnostic kit for the diagnosis of animal brucellosis in BPD, characterized in that it contains brucellosis antigen for BPD obtained according to any one of claims 9-10, brucellosis diagnostic lyophilized serum obtained according to any one of claims 12-19, and healthy blood serum cattle lyophilized. 22. A diagnostic kit for the diagnosis of animal brucellosis in KR with milk, characterized in that it contains brucellosis antigen for CR obtained according to claim 11 and brucellosis diagnostic serum lyophilized obtained according to any one of claims 12-19.