Method of producing antibody erythrocytic pasteurellosis diagnosticum

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

 

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

A method of obtaining or antibody-based test pasterilezom erythrocytic diagnosticum by processing the media antibody sera obtained on the antigens of pasteurellosis (Application RIGA for serological identification of pasteurellosis, Proceedings of all-Russian research Institute of veterinary Virology and Microbiology, goknow, 1998, str-366).

The disadvantages 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 pasterilezom erythrocytic diagnosticum by processing the media antibody sera obtained on the antigens of pasteurellosis, characterized in that as antigens pasteurellosis using capsular antigens derived from cultures of pasteurellosis by extraction of 2.0-2.5%sodium chloride solution at 40-42°C for 30-40 min, centrifugation of the extract, Department supernat the NTA and heating it at 65-70°C for 15-30 min with subsequent sterilizing filter.

The task is also solved in the method of obtaining or antibody-based test pasterilezom erythrocytic diagnosticum the fact that as antibodies sera using gamma-globulin fraction or immunoglobulin G or M

In the patent and scientific literature is not well-known technical 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 highly active antibodies on the most important immunogenic components of bacterial cell pasteurellosis - capsular antigens. In this regard, the development of stable and specific pastureland red blood cell or antibody-based test diagnostics resulting from sensitization of media antibody sera obtained from hyperimmunization animals highly specific capsular antigens of pasteurellosis is the most important task for display and serological identification. We first established that extraction pasterilezom capsular antigen from cultures of pasteurellosis solution of sodium chloride determined the military concentration at 40-42°C for 30-40 min, followed by heating it at 60-70°C for 15-30 min, used to hyperimmunization animals and antibodies, lets get the obvious positive effect of improving the quality of the target product at the expense of apparently reducing the presence of serologically related components from different serotypes of pasteurellosis in the composition of the capsular antigens, which leads to increased release of soluble capsular components 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.

Selected 16 hour broth culture of industrial strains of pasteurellosis: Pasteurella multocida /P. multocida/ serotypes A, B, D No. 1231, 681, T-80 and Pasteurella haemolytica biotype a-1, seeded in metrovia flask with 1.5%agar of Hottinger containing 10% serum of the horse, sterile, not inactivated, incubated at 37°C for 16 hours. Grown bacterial mass with a spatula washed off from the surface of the agar to 2.0%sodium chloride solution (pH 7,3), bring to 2.0%sodium chloride solution (pH of 7.3) the concentration of the resulting suspension to 40 billion M.L. (optical standard turbidity) and the extraction is carried out pasterilezom capsular antigen at 40°C for 30 min, then centrifuged at 10,000 rpm in those which begins 30 minutes and separate the supernatant. The obtained supernatant is heated at 70°C for 15 min, centrifuged at 15,000 rpm for 15 min and hold sterilizing filtration through millerovskie filters with membrane type GS-0,22.

The resulting supernatant used as capsular 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 /edited Chrimes/. Publishing house "Mir", M, 1979, str-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 pasterilezom 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 2 hours at 43°C With subsequent processing of 0.55%solution of neutral f is Malina. 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 pasterilezom erythrocytic diagnosticum check in the cross-TPHA with Homo - and heterologous capsular antigens of pasteurellosis. The activity of the obtained diagnosticum to Pasteurella multocida serotypes A, B, D, and Pasteurella haemolytica was 1:2400, 1:3200, 1:2400, 1:1800 respectively. The titer of antibody in a heterologous system interaction for the received diagnostics to Pasteurella multocida serotypes A, B, D, and Pasteurella haemolytica equal 1:2, 1:4, 1:4 and 1:8 respectively.

Example 2.

Selected 17 hour broth culture of industrial strains of pasteurellosis: Pasteurella multocida /P. multocida/ serotypes A, B, D No. 1231, 681, T-80 and Pasteurella haemolytica biotype a-1, seeded in flasks with 1.5%agar of Hottinger containing 10% serum of the horse, sterile, not inactivated, incubated at 37°C for 16 hours. Grown bacterial mass is extracted with a spatula from the surface of the agar with 2.5%sodium chloride solution (pH of 7.2) at 42°C for 40 min, adjusting the concentration of the resulting suspension to 40 billion M.L. (optical standard turbidity). The bacterial suspension is heated at 41°C for 30 min, centrifuged at 15000 rpm menu for 30 min and separated supernatant. The obtained supernatant is heated at 70°C for 15 min and hold sterilizing filtration through millerovskie filters with membrane type GS-0,22.

The resulting supernatant used as capsular 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 allocate fractions of the immunoglobulin G class known method (Immunological methods /edited Chrimes/. Publishing house "Mir", M, 1979, str-291). Fraction of immunoglobulin class G 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 pasterilezom erythrocytic diagnosticum. Processing media antibody carried out by mixing equal volumes of a 2.5%suspension formalisation Trinitarian erythrocytes horses and fractions of immunoglobulin class G 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. Activity poluinoplanetyanina of diagnosticum determine maximal titer in TPHA (reaction passive hemagglutination), using a set of specific homologous capsular antigens of pasteurellosis. Specificity or antibody-based test pasterilezom erythrocytic diagnosticum check in the cross-TPHA with Homo - and heterologous capsular antigens of pasteurellosis. The activity of the obtained diagnosticum to Pasteurella multocida serotypes A, B, D, and Pasteurella haemolytica was 1:3200, 1:2800, 1:2800, 1:1600 respectively. The titer of antibody in a heterologous system interaction for the received diagnostics to Pasteurella multocida serotypes A, B, D, and Pasteurella haemolytica equal 1:4, 1:4, 1:4 and 1:8 respectively.

Example 3.

Selected 18 hour broth culture of industrial strains of pasteurellosis: Pasteurella multocida /P. multocida/ serotypes A, B, D No. 1231, 681, T-80 and Pasteurella haemolytica biotype a-1, seeded in flasks with 1.5%agar of Hottinger containing 10% serum of the horse, sterile, not inactivated, incubated at 37°C for 16 hours. Grown bacterial mass is extracted with a spatula from the surface of the agar with 2.5%sodium chloride solution (pH of 7.2) at 42°C for 40 min, adjusting the concentration of the resulting suspension to 40 billion M.L.(optical standard turbidity). The bacterial suspension is heated at 41°C for 30 min, centrifuged at 15,000 rpm for 30 min and separated supernatant. The obtained supernatant is heated at 70°C for 15 min and hold sterilizing filtration is the situation through millerovskie filters with membrane type GS-0,22.

The resulting supernatant used as capsular 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 allocate fractions of the immunoglobulin M class known method of Immunological methods /edited Chrimes/. Publishing house "Mir", M, 1979, str-291). Fraction of immunoglobulin class M 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 pasterilezom erythrocytic diagnosticum. Processing media antibody carried out by mixing equal volumes of a 2.5%suspension formalisation Trinitarian erythrocytes horses and fractions of immunoglobulin class M 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 to the capsular antigens of pasteurellosis. Specificity or antibody-based test pasterilezom erythrocytic diagnosticum check in the cross-TPHA with Homo - and heterologous capsular antigens of pasteurellosis. The activity of the obtained diagnosticum to Pasteurella multocida serotypes A, B, D, and Pasteurella haemolytica was 1:2800, 1:2400, 1:2800, 1:1600 respectively. The titer of antibody in a heterologous system interaction for the received diagnostics to Pasteurella multocida serotypes A, B, D, and Pasteurella haemolytica equal 1:2, 1:4, 1:8 and 1:6 respectively.

Example 4.

Selected 16 hour broth culture of industrial strains of pasteurellosis: Pasteurella multocida /P. multocida/ serotypes A, B, D No. 1231, 681, T-80 and Pasteurella haemolytica biotype a-1, seeded in flasks with 1.5%agar of Hottinger containing 10% serum of the horse, sterile, not inactivated, incubated at 37°C for 16 hours. Grown bacterial mass is extracted with a spatula from the surface of the agar to 2.25%sodium chloride solution (pH of 7.3) at 41°C for 35 min, adjusting the concentration of the resulting suspension to 40 billion M.L.(optical standard turbidity). The bacterial suspension is heated at 41°C for 30 min, centrifuged at 15,000 rpm for 30 min and separated supernatant. The obtained supernatant is heated at 67,5°C for 20 min and hold sterilizing filtration through millerovskie filters with membrane type GS-0,22.

Received supernatant used as capsular antigens to obtain monospecific hyperimmune sera known method Magicnikon and other (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 /edited Chrimes/. Publishing house "Mir", M, 1979, str-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 pasterilezom 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 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 pasteurellosis. Specificity or antibody-based test pasterilezom erythrocytic diagnosticum check in the cross-TPHA with Homo - and heterologous capsulization pasteurellosis. The activity of the obtained diagnosticum to Pasteurella multocida serotypes A, B, D, and Pasteurella haemolytica was 1:2800, 1:2400, 1:2800, 1:1600 respectively. The titer of antibody in a heterologous system interaction for the received diagnostics to Pasteurella multocida serotypes A, B, D, and Pasteurella haemolytica equal 1:4, 1:8, 1:8 and 1:6 respectively.

Example 5.

Selected 17 hour broth culture of industrial strains of pasteurellosis: Pasteurella multocida /P. multocida/ serotypes A, B, D No. 1231, 681, T-80 and Pasteurella haemolytica biotype a-1, seeded in flasks with 1.5%agar of Hottinger containing 10% serum of the horse, sterile, not inactivated, incubated at 37°C for 16 hours. Grown bacterial mass is extracted with a spatula from the surface of the agar to 2.25%sodium chloride solution (pH of 7.3) at 41°C for 35 min, adjusting the concentration of the resulting suspension to 40 billion M.L. (optical standard turbidity). The bacterial suspension is heated at 41°C for 30 min, centrifuged at 15,000 rpm for 30 min and separated supernatant. The obtained supernatant is heated at 67,5°C for 20 min and hold sterilizing filtration through millerovskie filters with membrane type GS-0,22.

The resulting supernatant used as capsular antigens to obtain monospecific hyperimmune sera known method Magicnikon and others (proceedings of the Institute of Leningradskogo research Institute of epidemiology and Microbiology. Laster, 1976, t, p.34-37). From the received hyperimmune sera allocate fractions of the immunoglobulin G class known method (Immunological methods /edited Chrimes/. Publishing house "Mir", M, 1979, str-291). Fraction of immunoglobulin class G 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 pasterilezom erythrocytic diagnosticum. Processing media antibody carried out by mixing equal volumes of a 2.5%suspension formalisation Trinitarian erythrocytes horses and fractions of immunoglobulin class G 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 pasterilezom erythrocytic diagnosticum check in the cross-TPHA with Homo - and heterologous capsular antigens of pasteurellosis. Activity diagnostics received the mA to Pasteurella multocida serotypes A, B, D, and Pasteurella haemolytica was 1:2800, 1:3200, 1:2800, 1:1600 respectively. The titer of antibody in a heterologous system interaction for the received diagnostics to Pasteurella multocida serotypes A, B, D, and Pasteurella haemolytica equal 1:4, 1:4, 1:4 and 1:8 respectively.

Example 6.

Selected 18 hour broth culture of industrial strains of pasteurellosis: Pasteurella multocida /P. multocida/ serotypes A, B, D No. 1231, 681, T-80 and Pasteurella haemolytica biotype a-1, seeded in flasks with 1.5%agar of Hottinger containing 10% serum of the horse, sterile, not inactivated, incubated at 37°C for 16 hours. Grown bacterial mass is extracted with a spatula from the surface of the agar to 2.25%sodium chloride solution (pH of 7.3) at 41°C for 35 min, adjusting the concentration of the resulting suspension to 40 billion M.L.(optical standard turbidity). The bacterial suspension is heated at 41°C for 30 min, centrifuged at 15,000 rpm for 30 min and separated supernatant. The obtained supernatant is heated at 67,5°C for 20 min and hold sterilizing filtration through millerovskie filters with membrane type GS-0,22.

The resulting supernatant used as capsular 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, 196, t, p.34-37). From the received hyperimmune sera allocate fractions of the immunoglobulin M class known method of Immunological methods /edited Chrimes/. Publishing house "Mir", M, 1979, str-291). Fraction of immunoglobulin class M 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 pasterilezom erythrocytic diagnosticum. Processing media antibody carried out by mixing equal volumes of a 2.5%suspension formalisation Trinitarian erythrocytes horses and fractions of immunoglobulin class M 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 pasterilezom erythrocytic diagnosticum check in the cross-TPHA with Homo - and heterologous capsular antigens of pasteurellosis. The activity of the obtained diagnosticum to Pasteurella multcida serotypes A, B, D, and Pasteurella haemolytica was 1:2400, 1:3200, 1:2400, 1:1600 respectively. The titer of antibody in a heterologous system interaction for the received diagnostics to Pasteurella multocida serotypes A, B, D, and Pasteurella haemolytica equal 1:4, 1:2, 1:2 and 1:6 respectively.

Example 7.

Selected 16 hour broth culture of industrial strains of pasteurellosis: Pasteurella multocida /P. multocida/ serotypes A, B, D No. 1231, 681, T-80 and Pasteurella haemolytica biotype a-1, seeded in flasks with 1.5%agar of Hottinger containing 10% serum of the horse, sterile, not inactivated, incubated at 37°C for 16 hours. Grown bacterial mass is extracted with a spatula from the surface of the agar to 2.0%sodium chloride solution (pH 7.4) at 40°C for 30 min, adjusting the concentration of the resulting suspension to 40 billion M.L. (optical standard turbidity). The bacterial suspension is heated at 41°C for 30 min, centrifuged at 15,000 rpm for 30 min and separated supernatant. The obtained supernatant is heated at 65°C for 30 min and hold sterilizing filtration through millerovskie filters with membrane type GS-0,22.

The resulting supernatant used as capsular 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 /edited Chrimes/. Publishing house "Mir", M, 1979, str-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 pasterilezom 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 2 hours at 43°C With subsequent processing of 0.55%solution of neutral formalin. The activity received or antibody-based test diagnosticum was determined by limiting titer in TPHA (reaction passive hemagglutination), using a set of specific homologous capsular antigens of pasteurellosis. Specificity or antibody-based test pasterilezom erythrocytic diagnosticum check in the cross-TPHA with Homo - and heterologous capsular antigens of pasteurellosis. The activity of the obtained diagnosticum to Pasteurella multocida serotypes A, B, d, and Pasteurella haemolytica was 1:3200, 1:3200, 1:2400, 1:1600 respectively. The titer of antibody in a heterologous system interaction for the received diagnostics to Pasteurella multocida serotypes A, B, D, and Pasteurella haemolytica equal 1:4, 1:4, 1:4 and 1:6 respectively.

Example 8.

Selected 17 hour broth culture of industrial strains of pasteurellosis: Pasteurella multocida /P. multocida/ serotypes A, B, D No. 1231, 681, T-80 and Pasteurella haemolytica biotype a-1, seeded in flasks with 1.5%agar of Hottinger containing 10% serum of the horse, sterile, not inactivated, incubated at 37°C for 16 hours. Grown bacterial mass is extracted with a spatula from the surface of the agar to 2.0%sodium chloride solution (pH 7.4) at 40°C for 30 min, adjusting the concentration of the resulting suspension to 40 billion M.L. (optical standard turbidity). The bacterial suspension is heated at 41°C for 30 min, centrifuged at 15,000 rpm for 30 min and separated supernatant. The obtained supernatant is heated at 65°C for 30 min and hold sterilizing filtration through millerovskie filters with membrane type GS-0,22.

The obtained supernatant was used as capsular 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 guy who eremony sera allocate fractions of the immunoglobulin G class known method (Immunological methods /edited Chrimes/. Publishing house "Mir", M, 1979, str-291). Fraction of immunoglobulin class G 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 pasterilezom erythrocytic diagnosticum. Processing media antibody carried out by mixing equal volumes of a 2.5%suspension formalisation Trinitarian erythrocytes horses and fractions of immunoglobulin class G 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 pasterilezom erythrocytic diagnosticum check in the cross-TPHA with Homo - and heterologous capsular antigens of pasteurellosis. The activity of the obtained diagnosticum to Pasteurella multocida serotypes A, B, D, and Pasteurella haemolytica was 1:2800, 1:3200, 1:2800, 1:1800 respectively. The titer of antibody in a heterologous system interaction obtained for the of diagnosticums to Pasteurella multocida serotypes A, B, D, and Pasteurella haemolytica equal 1:4, 1:4, 1:4 and 1:6 respectively.

Example 9.

Selected 18 hour broth culture of industrial strains of pasteurellosis: Pasteurella multocida /P. multocida/ serotypes A, B, D No. 1231, 681, T-80 and Pasteurella haemolytica biotype a-1, seeded in flasks with 1.5%agar of Hottinger containing 10% serum of the horse, sterile, not inactivated, incubated at 37°C for 16 hours. Grown bacterial mass is extracted with a spatula from the surface of the agar to 2.0%sodium chloride solution (pH 7.4) at 40°C for 30 min, adjusting the concentration of the resulting suspension to 40 billion M.L. (optical standard turbidity). The bacterial suspension is heated at 41°C for 30 min, centrifuged at 15,000 rpm for 30 min and separated supernatant. The obtained supernatant is heated at 65°C for 30 min and hold sterilizing filtration through millerovskie filters with membrane type GS-0,22.

The resulting supernatant used as capsular 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 allocate fractions of the immunoglobulin M class known method of Immunological methods /edited Chrimes/. Publishing house "Mir", Moscow, 1979, str-291). Fraction of the immunoglobulin M class warmed up 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 pasterilezom erythrocytic diagnosticum. Processing media antibody carried out by mixing equal volumes of a 2.5%suspension formalisation Trinitarian erythrocytes horses and fractions of immunoglobulin class M 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 pasterilezom erythrocytic diagnosticum check in the cross-TPHA with Homo - and heterologous capsular antigens of pasteurellosis. The activity of the obtained diagnosticum to Pasteurella multocida serotypes A, B, D, and Pasteurella haemolytica was 1:2800, 1:2800, 1:2800, 1:1600 respectively. The titer of antibody in a heterologous system interaction for the received diagnostics to Pasteurella multoida serotypes A, B, D, and Pasteurella haemolytica equal 1:4, 1:4, 1:4 and 1:8 respectively.

The activity of diagnosticum to Pasteurella multocida serotypes A, B, D, and Pasteurella haemolytica, obtained in a known manner, was 1:1024, 1:2048, 1:2048, 1:900 respectively. The titer of antibody in a heterologous system interaction for the received diagnostics to Pasteurella multocida serotypes A, B, D, and Pasteurella haemolytica equal 1:32, 1:32, 1:32 and 1:64, respectively.

As shown by the experimental results, the proposed antigenic pasteurellaceae erythrocytic diagnosticums to Pasteurella multocida serotypes A, B, D, and Pasteurella haemolytica help to improve the quality of the target product by increasing its activity 1.7-2 times, and also to increase the specificity of her 2-8 times.

1. The method of obtaining or antibody-based test pasterilezom erythrocytic diagnosticum by processing the media antibody sera obtained on the antigens of pasteurellosis, characterized in that as antigens pasteurellosis using capsular antigens derived from cultures of pasteurellosis by extraction of 2.0-2.5%sodium chloride solution at 40-42°C for 30-40 min, centrifugation of the extract, the separation of the supernatant and heating it at 65-70°C for 15-30 min with subsequent sterilizing filter.

2. The method of obtaining or antibody-based test pasterilezom erythrocytic diagnosticum according to claim 1, characterized in that as the e antibodies sera using gamma-globulin fraction or immunoglobulin G or M



 

Same patents:

FIELD: veterinary science.

SUBSTANCE: method includes infecting the transplantable cell culture, cultivating the infected culture, settling the cells and cell detritus by centrifugation, preparing the cell lysate and freezing-defrosting. Herewith cell lysate is twice exposed to 12 mcm ultrasound within 1 minute every 1 min. After-centrifugation residual debris are refilled with distilled water, exposed to the same ultrasound. Supernatant fluids containing antigen are combined. Then antigen is double-activated with β-propiolactone in ratio 1:1000 at first at 37° constantly stirring, then β-propiolactone is introduced again in the same ratio, with exposition 18 hours and at temperature +4°C.

EFFECT: produced inactivated blutang antigen has high specificity and activity (tbl) and can be used for serodiagnosis in CFT, diffusion precipitation and EIA reactions.

1 tbl, 3 ex

FIELD: medicine; microbiology.

SUBSTANCE: invention concerns area of medical microbiology and can be used at carrying out of the laboratory control of the foodstuffs on contamination pathogenic biological agents in the conditions of extreme situations. In the invention the new technological scheme of carrying out of the laboratory control of the foodstuffs where preparation of assays spend to two stages is offered: the first stage - sample preparation - depending on density, fat content and fluidity of a product it is exposed, accordingly: to weighing, allocating 25 g (ml) of a product with the subsequent crushing, to transfer in a liquid phase in volume of 50-100 ml by means of normal saline solution addition, and at water research in number of 500 ml filter last through membranous filters; the second stage provides division of assay into parts: on 2 ml of assays separate for researches by methods of fluorescent antibodies (MFA), in the reaction of an indirect hemagglutination (RIHA), in reaction of a sintering of volume (RSV), enzyme immunoassay (EIA), polimerase chain reaction (PCR) and a bacteriological method and on 5 ml of assays take for research by a biological method, infecting biotrial animals. Research of assays by the express or accelerated methods (MFA, RIHA, RSV, EIA, PCR) spend within 4-10 hours, and research by a biological method - within 48-120 hours with use of selective nutrient mediums, such as the ADET-agar diagnostic elective tularemic, ADEA-agar diagnostic elective anthracic, EEDC-environment elective diagnostic choleraic, PYM-37 plague yeast medium.

EFFECT: unification of carrying out of the laboratory analysis of the foodstuff; parallel research of 25 assays, which preparation takes 1,5-2 hours.

6 cl, 10 tbl

FIELD: medicine.

SUBSTANCE: given invention concerns area of medicine and concerns the method of identification and extraction of endotoxins in the sample and their excisions from the sample. The essence of the invention includes the method of detection of an endotoxin of gramme-negative bacteria by means of an incubation of the sample with p12 or p12-like fiber of a bacteriophage, detection of the endotoxin bound to caudal fiber of a bacteriophage, thus a way of excision of an endotoxin includes an incubation of the sample with caudal fiber p12 a bacteriophage and excision of a complex from the sample.

EFFECT: simplification of the method of identification and extraction of endotoxins.

15 cl, 17 ex, 1 tbl 11 dwg

FIELD: medicine; veterinary medicine.

SUBSTANCE: method consists in fractional formalinisation of sheep erythrocytes and their sensitisation with mycoplasma antigen at 70°C during 30 minutes. The erythrocytes being used without prior tannage; they are loaded with sensitiser, obtained from proportional mix of M. bovoculi, M. argenini and Ureaplasma sp. cultures, and heated in water bath at 70°C during 30 minutes. The diagnosticum produced is washed three times, without adding 0.4% of normal rabbit serum on final stage.

EFFECT: high specificity and activity of indirect hemagglutination reaction.

2 tbl

FIELD: medicine, microbiology.

SUBSTANCE: invention concerns area of biotechnology and medical virology. The diagnostic test system for revealing of a virus of bird flu A/H5N1 is offered at enzyme immunoassay carrying out on a solidphase carrier with use of peroxidase conjugate. As a solidphase carrier the activated aluminosilicate matrix with a magnetic material with immobilized immunoglobulins against a virus of bird flu is used. The invention can be used for diagnostics of a virus of bird flu A/H5N1.

EFFECT: obtaining of diagnostic test system for revealing if virus of bird flu A/H5N1 at enzyme immunoassay carrying out on solidphase carrier with use of peroxidase conjugate.

FIELD: medicine; gastroenterology.

SUBSTANCE: perform microflora analysis, thus at detection as a part of an endoluminal jejunum microflora of concentration of a microflora of 107 microbic cells in 1 ml of intestinal contents and more and at concentration of a mucous microflora in biopsy materials of a jejunum of 1013 microbic bodies in 1 g of biopsy material and more and at content in biopsy material of mucosa of a jejunum and among an endoluminal microflora of a jejunum of eubacteria, lactic acid bacillus, aerobic actinomycetes, aerobic coccuses and anaerobia 75% and more from a total microbiocenosis of an intestine, and also at a phagocytic index less than 27% and level of secretory immunoglobulin IgA of 190 mkg/ml and less, prognosticate IBS development as a postinfectious syndrome.

EFFECT: increase of accuracy of predicting development of an irritable bowel syndrome.

2 ex

FIELD: medicine, veterinary.

SUBSTANCE: invention relates to the field of veterinary and concerns causative agents of new bacterial disease of poultry Pasteurella trehalosi and/or Mannheimia haemolytica. The claim describes bacteria Pasteurella trehalosi and Mannheimia haemolytica, causing diseases of upper respiratory and genital tracts in poultry. The said inactivated or alive weakened bacteria can be used for preparing therapeutical compositions against causative agents of the said poultry diseases. Invention relates to vaccines for prevention of the aforesaid diseases containing inactivated and/or alive weakened bacteria Pasteurella trehalosi and/or Mannheimia haemolytica, as well as to the method of immunisation in order to prevent the said disease in chickens using vaccine in immunologically effective amount. Invention allows for identifying new bacterial disease of poultry, detecting causative agent of disease and preventing outbreak of disease by means of elaborated vaccine.

EFFECT: possibility to identify new bacterial disease of poultry, detect causative agent of disease and prevent outbreak of disease by means of elaborated vaccine.

9 cl, 22 dwg, 5 tbl, 6 ex

FIELD: medicine; oncology.

SUBSTANCE: additionally cell cytoplasm labeling indices are defined for each Bc1-2 and p53 albumen. Tissue sample is taken from the tumour cut out during operation, tumour sections for immunohistochemical analysis are prepared. At the tomour sections expression of Ki-67, Bc1-2 and p53 albumens in cell nuclei is registered by specific monoclonal antibodies in a complex with chromogen. For each albumen, cell nucleus labeling index is defined by the following procedure: average percentage of cells with nucleus coloured by the chromogens are registered over more than 30 visual fields in histological sections processed by one of the specific monoclonal antibodies. Additionally cell cytoplasm labeling indices are defined by the same procedure for Bc1-2 and p53 albumens, potential probability index for proliferation activity (PPIPA) is evaluated by the formula: PPIPA=(LInucBc1-2/LInucp53)•LInucKi-67, where LInucBc1-2, LInucp53, LInucKi-67 are cell nucleus labeling indices for each of the Bc1-2, p53 or Ki-67 albumens respectively; possible cytoplasmic atypia index (PAI) is evaluated as a ratio of cell cytoplasm labeling index of Bcl-2 albumen to cell cytoplasm labeling index for p53 albumen; tumour growth and potential malignancy index (TGPMI) is defined as a product of PAI and PPIPA; at 0≤TGPMI≤4.35 atypical follicular adenoma is indentified, and at TGPMI>4.36 follicular cancer is identified.

EFFECT: increased accuracy of post-operational differential diagnostics of follicular adenoma and follicular cancer in thyroid gland.

4 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, specifically to immunology and concerns method of antibodies test to capsular polysaccharide Haemophillus influenzae type b. Substance of invention implies that working cultivations of analysed and reference serum are applied on solid binder with fixed high-purity capsular polysaccharide Haemophilus influenzae type b, as well as marker samples referring as to serums with relative content of IgG antibodies to capsular polysaccharide polyribosylribitolephoaphate Haemophilus influenzae are incubated, isolated from surplus antibodies, added with conjugate in working cultivation, repeatedly incubated at the same parametres, washed from surplus conjugate, added with substrate mixture containing tetramethylbenzidine and hydrogen peroxide in preset values, incubated in the lightproof place. Reagent is added to stop reaction; optical density of analysed and reference serum are tested for antibodies content. Diagram optical density and conditional content of antibodies in marker samples is drawn and used to detect antibodies content in analysed samples, in standard units. Advantage of invention consists in increase of method sensitivity.

EFFECT: increased sensitivity of method of antibodies test to capsular polysaccharide Haemophillus influenzae type b.

2 tbl, 1 dwg

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to immunology, and can be used for testing of postvaccinal immunity within vaccination by vaccine "Pneumo-23". Substance of invention consists that solid carrier with sorbate vaccine " Pneumo-23", containing 23 pneumococcus polysaccharides is covered with working cultivations of analysed and reference serum, incubated at temperature 18-22°C within 30 minutes, isolated from surplus antibodies, added with conjugate of working cultivation, repeatedly incubated at the same parameters, washed from surplus conjugate, added with substrate mixture containing tetramethylbenzidine and hydrogen peroxide of specified values, incubated at temperature 18-22°C within 15-20 minutes in lightproof place protected. Reagent is added to stop reaction. Optical density of analysed and reference serum are evaluated for antibody content prior to and after vaccination. Optical density of analysed serum increased in 4 times and more indicates postvaccinal immunity. Advantage of invention consists in increase of method sensitivity.

EFFECT: increased sensitivity of method of postvaccinal immunity test within vaccination by vaccine "Pneumo-23".

1 tbl

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 antigen erythrocytic colibacillosis diagnosticum by extracting an adhesive antigen from Escherichia cultures, centrifuging the extract, and separating the supernatant. Thereafter formalinised tanninised animal erythrocytes are sensitised with the produced antigen. Extraction is performed in culture fluid containing Escherichia cell culture 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. After extraction the supernatant is heated up at 65-68°C within 25-30 min.

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

3 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 antigen erythrocytic pasteurellosis diagnosticum by extracting pasteurellosis capsular antigen from Pasteurella cultures with sodium chloride brine, with centrifuging the extract and separating the supernatant exposed to sterilisation filtration and used for sensitisation of formalinised anionised animal erythrocytes. Pasteurellosis capsular antigen is extracted with 2.0-2.5% sodium chloride brine at 40-42°C during 30-40 min. Prior to sterilisation filtration, the supernatant is heated up at 65-70°C within 15-30 minutes.

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

3 ex

FIELD: medicine.

SUBSTANCE: invention refers to virology, namely, to methods of immunosorbent production. Method of immunosorbent production virus-specific antibody binding is offered. Method includes inorganic sorbent-carrier incubation with virus-containing liquid. Ultradisperse oxygen-containing graphite is used for inorganic sorbent-carrier as foamed particles of the stratified graphite, containing oxygen in amount 12-14 mass % per 73-76 mass % of carbon. Specific surface of graphite is 1500-2000 m3/g, particle size is 25-50 mcm. Graphite is pre-boiled in distilled water. Produced suspension of sorbent-carrier at graphite content not less than 2 mass % is incubated with virus-containing liquid at temperature 5-35°C. Virus-containing liquid contains, e.g. influenza virus.

EFFECT: produced immunosorbent for virus-specific antibody binding has high sorptive power, enables to bind completely virus-specific antibodies of immune serum.

2 cl, 5 tbl

FIELD: medicine; veterinary science.

SUBSTANCE: produced plasma after it is separated from blood and refined from trypanosome deposition is processed with 20-25% polyethylene glycol solution taken in proportion equal to blood plasma volume. Then produced mixture is kept at room temperature for 12-15 min, recentrifuged at 6000 revolutions/min for 15-20 min, after that supernatant is removed, and produced deposition is used as trypanosome exoantigen for serological reaction. At that its activity should be 95-97%.

EFFECT: timely finding of sick animals and possibility to take emergency measures for invasion elimination.

2 tbl

FIELD: medicine; gastroenterology.

SUBSTANCE: cell lysate is received by double destruction of bacterial cells: first, treatment with 1% sodium desoxycholate solution at a rate of 0.5 ml solution to 0.2 ml suspension with concentration (5-7) × 1010 microbes/ml, with following stirring at magnetic agitator in thermostat at 40°С for 2 hours, second, by performing 5 per 45 sec cycles of ultrasound disintegration, after which, the obtained suspension is precipitated by centrifuging at 8000 rpm for 40 min, and the obtained cell lysate is used as a sensitin, which is immobilised at polymer carrier with the following lyophilisation. In sensitin, the protein concentration 1.5-2 mg/m with wide immunoglobulin spectrum is received. As sensitin carrier, the globular polymeric particles with diameter 1.5 mcm and containing 1.3 mmol/g aldehyde groups can be used, and the lysate-produced immobilisation of micro spheres is performed by use of 0.1 mol carbonate buffer with pH 9.2. Interlocking of free aldehyde groups is performed by adding 2.0 ml 0.5% gelatose on 0.9% sodium chloride to suspension with carrier and soluble antigen, and leave to stay at constant stirring at the agitator at 20°С fro 120 min.

EFFECT: method provides the quantitative determination of antibodies to HPylory and efficient application for controlling the eradicative therapy.

2 tbl, 2 ex, 4 cl

FIELD: medicine, veterinary.

SUBSTANCE: specific antibodies to virus of goose enteritis are determined by IEA where virus received following injection of epizootic strain "P-75" of enteritis virus, is used as an antigen, and macro porous glass with diameter not less than 700 is used for purification of virus-containing material, the goose Ig G specific immunoperoxidase conjugate is used as an anti-specific agent, and the IEA results are calculated by use of the formula: titer = antiIg[2.02(lg S/P)+3.51], where S is for optical density of tested serum; P is for optical density of the positive control.

EFFECT: method reduces the test time and economical costs.

2 tbl, 2 ex

FIELD: medical engineering.

SUBSTANCE: device has substrate having polymeric working layer on it, produced from copolymer based on methacrylic acid derivatives with biological macromolecules (probes) immobilized thereon. The substrate is manufactured from activated or not activated glass, metal or polymer material. The working layer has macroporous monolithic copolymer glycidyl methacrylate and ethylene glycol methacrylate taken in (50:70)-(50:30) proportions by mass with affine biological probes immobilized thereon. Probe-copolymer proportion is 2-10 mg/g of copolymer, for protein, 1-20 mg/g of copolymer for peptide and for oligonucleotide, nucleic acid - 0.5-3 mg/g of copolymer, pore radius of 0.4-1.5 mcm, it has thickness of 50-700 microns and is manufactured as continuous or discrete microcellular layer. The method for manufacturing biochip involves preparing substrate, producing working layer by monomer copolymerization on methacrylic acid derivatives base, immobilizing biological macromolecules - probes on forming copolymer, washing, drying the received biochip. Radical copolymerization of glycidyl methacrylate and ethylene glycol methacrylate taken in (50:70)-(50:30) proportions by mass is carried out for producing working layer with photo-or thermal initiation in poregenic solvent medium being applied. Proportion of the sum of monomer volumes to solvent volume being equal to 6:9, initiator concentration in reactionary medium being equal to 0.2-1.0% by weight, given reaction mixture is placed on substrate as continuous or discrete layer. Macroporous monolithic continuous or discrete microcellular layer is formed as a result of copolymerization on the substrate. Then, covalent immobilization of biological macromolecules is carried out in the layer pores or their direct synthesis on formed copolymer with its native or modified epoxy groups being used. Biological affine probe is produced. The probe is introduced into copolymer in quantity of 2-10 mg/g of copolymer for fiber, for peptide - 1-20 mg/g of copolymer and for oligonucleotide or nucleic acid - 0.5-3 mg/g of copolymer.

EFFECT: manufacturing reusable biochip with predetermined controllable and reproduced quality.

17 cl

FIELD: veterinary virology, in particular production of latex diagnosticum for diagnosis of horned cattle and poultry infections.

SUBSTANCE: claimed method includes centrifugation of polymeric suspension and adjusting of polymeric suspension with distilled water up to 0.5 % (calculated as polymer dry residue)/ Then equal volumes of viral antigen in infective titer of 6.5 lg TCD/50 ml (tissue cytopatic doses) and 0.5 %-1.0 % polymeric suspension are mixed and mixture is hold in thermostatic regulator at 36-38°C for 4-6 h. Further 0.07-0.15 % aqueous solution of human serum albumin is added into total suspension volume, mixture is incubated .at 4-8°C for 11-13 h; suspension is washed, and diagnosticum concentration is adjusted with phosphate buffered saline with pH 7.2-7.4 up to 0.1-0.3 %. Prepared diagnosticum is stored at 4°C not more than 1 year.

EFFECT: diagnosticum for before-the-fact detection of different infective diseases, prophylaxis and treatment.

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: biotechnology, medicine, immunology.

SUBSTANCE: method involves preparing control samples by preparing solution of heterologous chimeric antibodies consisting of whole molecules or fragments of immunoglobulin isolated from immunized animal serum and associated with whole molecules or fragments of human immunoglobulin in phosphate-saline buffer, pH 6.0 followed by preparing different dilutions in indicated buffer, their control in IFA and selection of dilutions at optical density values 1.0, not less. Invention provides preparing control samples comprising specific antibodies that elicit high specificity and capacity for detection in combination with their infectious safety, standard indices and availability with respect to economy aspects.

EFFECT: improved preparing method.

3 cl

FIELD: medicine.

SUBSTANCE: invention concerns area of medicine and concerns preparations, the antibody-containing person, applied to treatment of microbacterial infections. The essence of the invention includes the preparations containing human antibodies IgG either secretory IgA, or their admixture, specific to antigens Mycobacterium tuberculosis and the vaccine of Calmette and Guerin in concentration from 40 to 160 mg/ml and suitable pharmaceutical medium.

EFFECT: advantage of the invention consists in scope expansion.

5 cl, 2 ex, 3 tbl, 3 dwg

Up!