Method of producing antibody erythrocytic colibacillosis diagnosticum

FIELD: veterinary science.

SUBSTANCE: invention refers to veterinary microbiology and biotechnology can be used for development of specific colibacillosis diagnostic aids. According to the invention the method covers producing antibody erythrocytic colibacillosis diagnosticum and provides carrier processing with antibodies to Escherichia antigens being adhesive antigens extracted from Escherichia cultures with phosphate-carbamide buffer 1.8-2.0 M of pH 7.2-7.4 at temperature 40-45°C during 25-30 min. The culture fluid containing Escherichia cell culture and phosphate-carbamide buffer are taken in mass ratio 1:0.4-0.6 respectively. The supernatant from the extraction is heated up at 65-68°C within 25-30 min. Gamma globulin fractions are used as serum antibodies for carrier processing.

EFFECT: method allows for high-quality end product due to improved sensitivity and specificity.

3 ex

 

The invention relates to veterinary Microbiology and biotechnology and can be used in the development of specific diagnostics and, in particular, to obtain or antibody-based test esherihioznae erythrocytic diagnosticum.

A method of obtaining or antibody-based test esherihioznae erythrocytic diagnosticum by processing the media by antibodies raised to antigens of Escherichia ("Determination of adhesion antigens from pathogenic Escherichia and detection of anti-adhesive antibodies in TPHA", Environmental aspects of the epizootiology and pathology of animals, international science and production conference, dedicated to the 100th anniversary since the birth Kotova V.T., Voronezh, 1999, s-178).

The disadvantage of diagnosticum is the low quality of the target product, characterized by its low sensitivity and specificity.

Objective of the claimed technical solution is to improve the quality of the target product by increasing its sensitivity and specificity.

The problem is solved in the method of obtaining or antibody-based test esherihioznae erythrocytic diagnosticum by processing the media by antibodies raised to antigens of Escherichia fact that as antigens of Escherichia use adhesive antigens derived from cultures of Escherichia by extraction, 1,8-2,0 M FOSFA the IDT-urea buffer with a pH of 7.2 to 7.4 at a temperature of 40-45°C for 25-30 min, moreover, the culture fluid containing the cell culture of Escherichia, and phosphate-urea buffer taken in a mass ratio of 1:0.4 to 0.6, respectively, and the supernatant after extraction heated at 65-68°C for 25-30 min, whereas the antibodies of sera for processing media using the gamma-globulin fraction.

In the patent and scientific literature unknown solutions containing elements similar to the claimed, i.e. the proposal meets the criterion of "novelty".

The proposal was feasible in laboratory and industrial conditions, aimed at solving real technical problem, i.e. the sentence "industrially applicable".

During the development of red blood cell or antibody-based test diagnostics is of paramount importance to obtain active antibodies on the most important immunogenic components of bacterial cells of Escherichia. In this regard, the development of stable and specific exericising erythrocytic diagnosticums, the resultant hyperimmunization animals highly specific adhesive antigens of Escherichia, is an important task for display and serological identification.

We first established that extraction esherihioznae adhesive antigen from cultures of Escherichia directly into the culture liquid containing the culture to etoc Escherichia, 1,8-2,0 M phosphate-urea buffer with a pH of 7.2 to 7.4 at a temperature of 40-45°C for a certain time, and the ratio of the culture liquid containing the cell culture of Escherichia, and phosphate-urea buffer taken in a mass ratio of 1:0.4 to 0.6, respectively, and the supernatant after extraction of heat under certain temperature conditions, leading to increased release of soluble adhesive component in the extract and to improve the sensitivity and specificity or antibody-based test diagnosticum, i.e. the proposal meets the criteria of "novelty" and "inventive step".

The method is illustrated by the following examples.

Example 1.

Adhesive antigens To ab 88, 88 AC, 88 ad, C, F-41, Att-25, R E. coli was prepared as follows. Culture industrial strains of Escherichia E. coli specified adhesive antigens (strains No. 729, 661/16, 798, 957, 688/19, 4 and 745) grown in a broth of Hottinger. Each strain is grown separately for 18 h at 37°C. the Grown strains separated from the culture fluid by centrifugation at 10,000 rpm for 10 min. Culture liquid is poured into a separate container. In the obtained precipitate after centrifugation grown crops determine the concentration of microbial cells and bring it to a concentration of 150 billion microbial cells/ml of culture fluid. Extraction adhesive anti-Christ. ENES Escherichia carried out directly in the culture fluid of 1.8 M phosphate-urea buffer with a pH of 7.2 for 25 min at 40°C, moreover, the culture fluid containing the cell culture of Escherichia, and phosphate-urea buffer taken in a mass ratio of 1:0.4 respectively. Then the reaction liquid is centrifuged at 10,000 rpm for 20 minutes the resulting supernatant is heated at 65°C for 25 min, cooled to 5°C and re-centrifuged 10,000 rpm for 20 minutes the resulting supernatant used as the adhesive antigens to obtain monospecific hyperimmune sera known method Magicnikon and others (proceedings of the Institute of the Leningrad scientific research Institute of epidemiology and Microbiology. Laster, 1976, t, p.34-37). From the received hyperimmune sera emit gamma-globulin fraction known method (Immunological methods. Ed. Chrimes/. M., Mir, 1979, s-291). The fraction of gamma-globulin were heated for 1 hour at 56°C, followed by cooling to 0°C for 15 min and used as antibodies for processing the medium and cook or antibody-based test esherihioznae erythrocytic diagnosticum. Processing media antibody carried out by mixing equal volumes of a 2.5%suspension formalisation Trinitarian erythrocytes horses and fractions immune gamma globulin in a known manner according to the method of levy M.I. and others (In kN.: "Materials of the 3rd International conference of institutions of vaccines and sera, social the socialist countries", M., 1965, p.32).

The mixture is heated for 2 hours at 43°C With subsequent processing of 0.55%solution of neutral formalin. The activity received or antibody-based test diagnosticum determine maximal titer in TPHA (reaction passive hemagglutination), using a set of specific homologous capsular antigens of Escherichia. Specificity or antibody-based test esherihioznae erythrocytic diagnosticum check in the cross-TPHA with Homo - and heterologous adhesive antigens of Escherichia. The activity of the obtained diagnosticum for E. coli (strains No. 729, 661/16, 798, 957, 688/19, 4 and 745) was 1:1200, 1:1200, 1:2400, 1:1800, 1:1600, 1:2400, 1:1200 respectively. The titer of antibody in a heterologous system interaction for the received diagnostic for E. coli (strains No. 729, 661/16, 798, 957, 688/19, 4 and 745) equal 1:2, 1:2, 1:2, 1:2, 1:2, 1:2, 1:2 respectively.

Example 2.

Adhesive antigens To ab 88, 88 AC, 88 ad, C, F-41, Att-25, R E. coli was prepared as follows. Culture industrial strains of Escherichia E. coli specified adhesive antigens (strains No. 729, 661/16, 798, 957, 688/19, 4 and 745) grown in a broth of Hottinger. Each strain is grown separately for 20 h at of 36.5°C. the Grown strains separated from the culture fluid by centrifugation at 15,000 rpm for 15 minutes Culture liquid is poured into a separate container. In the obtained precipitate after centrifugation exp is illicit crops determine the concentration of microbial cells and bring it to a concentration of 150 billion microbial cells/ml of culture fluid. Extraction of the adhesive antigens of Escherichia spend directly from crop production strains of Escherichia in culture liquid of 2.0 M phosphate-urea buffer with pH 7.4 for 30 min at 45°C, and the culture fluid containing the cell culture of Escherichia, and phosphate-urea buffer taken in a mass ratio of 1:0.6 to. Then the reaction liquid is centrifuged at 18000 rpm for 30 minutes the resulting supernatant is heated at 68°C for 30 min, cooled to 0°C and re-centrifuged 15,000 rpm for 15 minutes, the resulting supernatant is used as the adhesive antigens to obtain monospecific hyperimmune sera known method Magicnikon and others (proceedings of the Institute of the Leningrad scientific research Institute of epidemiology and Microbiology. Laster, 1976, t, p.34-37). From the received hyperimmune sera emit gamma-globulin fraction known method (Immunological methods. Ed. Chrimes/. M., Mir, 1979, s-291). The fraction of gamma-globulin were heated for 1 hour at 56°C, followed by cooling to 0°C for 15 min and used as antibodies for processing the medium and cook or antibody-based test esherihioznae erythrocytic diagnosticum. Processing media antibody carried out by mixing equal volumes of a 2.5%suspension of formalisation the x Trinitarian erythrocytes horses and fractions immune gamma globulin in a known manner according to the method of levy M.I. and others (In kN.: "Materials of the 3rd International conference of institutions of vaccines and sera, the socialist countries", M., 1965, p.32).

The mixture is heated for about 1.75 hours at 44°C With subsequent processing of 0.65%solution of neutral formalin. The activity received or antibody-based test diagnosticum determine maximal titer in TPHA (reaction passive hemagglutination), using a set of specific homologous capsular antigens of pasteurellosis.

Specificity or antibody-based test esherihioznae erythrocytic diagnosticum check in the cross-TPHA with Homo - and heterologous adhesive antigens of Escherichia. The activity of the obtained diagnosticum for E. coli (strains No. 729, 661/16, 798, 957, 688/19, 4 and 745) was 1:1200, 1:1200, 1:2400, 1:1800, 1:1600, 1:2400, 1:1200 respectively. The titer of antibody in a heterologous system interaction for the received diagnostic for E. coli (strains No. 729, 661/16, 798, 957, 688/19, 4 and 745) equal 1:2, 1:2, 1:2, 1:2, 1:2, 1:2, 1:2 respectively.

Example 3.

Adhesive antigens To ab 88, 88 AC, 88 ad, C, F-41, Att-25, R E. coli was prepared as follows. Culture industrial strains of Escherichia E. coli specified adhesive antigens (strains No. 729, 661/16, 798, 957, 688/19, 4 and 745) grown in a broth of Hottinger. Each strain is grown separately for 20 h at of 36.5°C. the Grown strains separated from the culture fluid by centrifugation at 15000 rpm for the tion 15 minutes The culture fluid is drained into a separate container. In the obtained precipitate after centrifugation grown crops determine the concentration of microbial cells and bring it to a concentration of 150 billion microbial cells/ml of culture fluid. Extraction of the adhesive antigens of Escherichia spend directly from crop production strains of Escherichia in the culture fluid of 1.9 M phosphate-urea buffer with a pH of 7.3 for 28 min at a temperature of 42.5°C, and the culture fluid containing the cell culture of Escherichia, and phosphate-urea buffer taken in a mass ratio of 1:0.6 to. Then the reaction liquid is centrifuged at 14000 rpm for 25 min Obtained supernatant is heated at 66,5°C for 28 min, cooled to 3°C and re-centrifuged 18000 rpm for 10 minutes the resulting supernatant used as the adhesive antigens to obtain monospecific hyperimmune sera known method Magicnikon and others (proceedings of the Institute of the Leningrad scientific research Institute of epidemiology and Microbiology. Laster, 1976, t, p.34-37). From the received hyperimmune sera emit gamma-globulin fraction known method (Immunological methods. Ed. Chrimes/. M., Mir, 1979, s-291). The fraction of gamma-globulin were heated for 1 hour at 56°C, followed by cooling to 0°C for 15 mi is used as antibodies for processing the medium and cook or antibody-based test esherihioznae erythrocytic diagnosticum. Processing media antibody carried out by mixing equal volumes of a 2.5%suspension formalisation Trinitarian erythrocytes horses and fractions immune gamma globulin in a known manner according to the method of levy M.I. and others (In kN.: "Materials of the 3rd International conference of institutions of vaccines and sera, the socialist countries", M., 1965, p.32).

The mixture is heated for 1.5 hours at 45°C With subsequent processing of 0.6%solution of neutral formalin. The activity received or antibody-based test diagnosticum determine maximal titer in TPHA (reaction passive hemagglutination), using a set of specific homologous capsular antigens of pasteurellosis. Specificity or antibody-based test esherihioznae erythrocytic diagnosticum check in the cross-TPHA with Homo - and heterologous adhesive antigens of Escherichia. The activity of the obtained diagnosticum for E. coli (strains No. 729, 661/16, 798, 957, 688/19, 4 and 745) was 1:1200, 1:1200, 1:2400, 1:1800, 1:1600, 1:2400, 1:1200 respectively. The titer of antibody in a heterologous system interaction for the received diagnostic for E. coli (strains No. 729, 661/16, 798, 957, 688/19, 4 and 745) equal 1:2, 1:2, 1:2, 1:2, 1:2, 1:2, 1:2 respectively.

The activity of diagnosticum for E. coli (strains No. 729, 661/16, 798, 957, 688/19, 4 and 745), obtained in a known manner, was 1:400, 1:800, 1:600, 1:800, 1:400, 1:800, 1:600 respectively. The titer of antibody in a heterologous si is the subject of the interaction obtained for diagnostic E. coli (strains No. 729, 661/16, 798, 957, 688/19, 4 and 745) equal 1:6, 1:8, 1:8, 1:6, 1:8,1:8 1:12, respectively.

As shown by the experimental results, the proposed or antibody-based test exericise erythrocytic diagnosticums for E. coli (strains No. 729, 661/16, 798, 957, 688/19, 4 and 745) help to improve the quality of the target product by increasing its activity 1.5-4 times, and also to increase the specificity of its 2-6 times.

The method of obtaining or antibody-based test esherihioznae erythrocytic diagnosticum by processing the media by antibodies raised to antigens of Escherichia notable as antigens of Escherichia use adhesive antigens derived from cultures of Escherichia by extraction, 1,8-2,0 M phosphate-urea buffer with a pH of 7.2 to 7.4 at a temperature of 40-45°C for 25-30 min, and the culture fluid containing the cell culture of Escherichia, and phosphate-urea buffer taken in a mass ratio of 1:0.4 to 0.6, respectively, and the supernatant after extraction heated at 65-68°C for 25-30 min, and the serum antibodies for processing media using the gamma-globulin fraction.



 

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7 ex, 7 tbl

FIELD: veterinary microbiology.

SUBSTANCE: claimed method includes providing of stable culture from B.abortus 19 strain in L-form followed by rabbit immunization. Culture is obtained in dense broth containing additionally 10-15 % of normal hoarse serum wherein broth is singly exposed to streptomycin action in dose of 2.5-5.0 U/ml of medium. Then slurry is prepared from obtained culture, inactivated at 85-90°C for 160 min and triply intravenously administrated to rabbits in increasing doses with 72 h intervals.

EFFECT: method for more exact estimation of brucelliasis epizootic situation on basis of typical, dissociated and deep-altered brucella forms.

6 tbl, 4 ex

FIELD: veterinary science.

SUBSTANCE: invention refers to veterinary microbiology and biotechnology, can be used for development of specific diagnostic aids. According to the invention the method covers producing antibody erythrocytic pasteurellosis diagnosticum by carrier processing with serum antibodies of Pasteurella antigens being capsular antigens extracted from Pasteurella cultures with 2.0-2.5% sodium chloride brine at 40-42°C during 30-40 min, with centrifuging the extract, separating the supernatant thereafter warmed up at 65-70°C during 15-30 min followed with sterilisation filtration. The serum antibodies for making diagnosticum are gamma globulin fraction or G or M immunoglobulins.

EFFECT: application of the method allows for high-quality end product due to improved sensitivity and specificity.

2 cl, 9 ex

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