Method for differential diagnostics of bovine brucellosis and a method of preparing a preparation for implementation thereof

FIELD: veterinary and medicine.

SUBSTANCE: invention, in particular, relates to production and use of biological preparations intended for differential diagnostics of brucellosis and to a method of differentially diagnosing brucellosis. Method involves serologic analysis of sera using antigenic "IFK", which is horse-radish peroxidase-labeled electrophoretically purified polypeptide fraction of virulent strain B.arborus 54. Diagnosis of brucellosis is stated when anti-brucellosis antibodies in sick animal sera diluted to 1/100 and higher are revealed at CSP reaction intensity 2.1 and higher.

EFFECT: elaborated method increasing immuno-enzymatic test specificity and allowing performing differentiation of postvaccinal immunological reactions and postinfectious reactions induced by microorganisms having antigenic affinity with brucellas, especially Yersinia enteriocolitica.

2 cl, 4 ex

 

The invention relates to veterinary medicine, in particular the production and manufacture of drugs intended for the differential diagnosis of brucellosis.

There are several ways serological and differential diagnosis of brucellosis in cattle. It is such as agglutination reaction (RA), the reaction of complement fixation (RAC), long reaction of complement fixation (GSK), the reaction of indirect haemagglutination (rnga), rose Bengal test (BPO) and others.

The disadvantage of these methods is the lack of strict specificity of these test systems, because in many cases the animals are identified reactions that are not associated with the development of brucellosis infectious process. This is because as the primary immunological component in these test systems is a single Brucella antigen that reacts with S-RS-antibodies that are present both in patients with brucellosis and grafted protivopoloznymi vaccines, as well as in animals - carriers of Yersinia - Y.enterocolitica.

The closest technical solution, selected as a prototype, is the ELISA (enzyme-linked immunosorbent assay), which for the differential diagnosis of brucellosis conduct serological indication of Brucella in the interest of veterinary surveillance using monoclonal and the antibodies (ICA). In sensitized Brucella antigen tablets make monoclonal antibodies, and after holding at a temperature of 37-38°C for 1 hour add individuai enzyme conjugate. About the presence of antigens in the test sample is judged by the intensity of the color content of the holes. The reaction is considered positive when the intensity of staining 2-4 cross in dilutions 1/16-1/32. (Plotnikova AM, Salmanov K.M. - the Development of methods and means of immunomonitoring brucellosis in animals. - Veterinary. - 2003. No. 12. - P.16-18.)

However, differential diagnosis of brucellosis using ELISA test by indicating the causative agent of brucellosis (Brucella antigen) associated with technological difficulties, for the process agent indication is very time consuming and difficult task, because for detection of the pathogen is necessary to prepare samples for the study, the accumulation of bacterial biomass, the processing of antigenic material and production ELISA.

The disadvantage of this method is the complexity and high cost of obtaining µa for ELISA, long and multi-stage preparation for the study, the accumulation of bacterial biomass processing antigenaemia material, etc. With the search agent may not be allocated as a result of preliminary bacteriological ind the requirements with a small number of microbes in the samples (less than 10 4microbial cells in 1 g (ml) of sample) can be negative, which reduces the sensitivity and efficiency for the serological diagnosis of brucellosis. In this way not achieved the main goal - differentiation of virulent strains of the pathogen from avirulent vaccine and variants of Brucella, which is associated with antigen R-forms of Brucella to hyperimmunization animal donor specific antibodies.

The objective of the invention is to develop a method that improves the specificity of the ELISA test and enables the differentiation of vaccine-induced immunological reactions from post-infectious, and differentiation cross-reactions induced by microorganisms with antigenic relationship with Brucella, especially Yersinia entericolica.

The problem is solved through the use of serological analysis of serum samples examined animals in the enzyme-linked immunosorbent assay (ELISA), which is carried out with the use of antigenic variant enzyme conjugate (AG IFC), which as a specific indicator of the component using the polypeptide fraction virulent strain of B.abortus 54 with a molecular weight of 41.8 kDa, missing the vaccine (B.abortus received 19.82, R-1096) and other virulent species (B. suis, B. obis) Brucella, and the phenomenon of enzyme reaction is judged on the presence of specifics is their post-infectious antibodies in the test sera, in terms of the intensity of the reaction (PSC) and post-infectious titer of antibodies in ELISA put the differential diagnosis for brucellosis, for diagnostic post-infectious titer of antibodies take positive in dilutions analyzed sera 1:100 and above with a factor specificity (PSC)of-2.1 and above.

The invention consists also in the fact that the method of obtaining antigenic variant enzyme conjugate (AG IFC) for the differentiation of post-infectious antibodies from vaccination includes radioactively Brucella subsequent legirovaniem them, the allocation of specific antigenic fractions of a virulent strain of B.abortus 54 electrophoretic method in polyacrylamide gel (PAG) in the concentration gradient of 10-20% in the presence of Na dodecyl sulfate (SDS), the force of the current of 80 mA and exposure of 2.5-3.0 hours, the fixation plates of the gel in a solution containing 40% isopropyle alcohol and 10% acetic acid, followed by staining of the gel in 0.1% solution of Kumasi diamond blue R-250, additional staining polypeptide fractions silver using a solution of formaldehyde, determination of molecular mass fractionated polypeptides using a calibration curve based on standard markers: BSA (CD), EA (CA) and cytochrome C (12,3 kDa), transfer of antigenic fractions with plastipak on nitrocellulose membrane ("Miliipore") buffer, containing 0,024 M Tris, 0,076 M glycine and 20% methanol using graphite plates (3 hours at 100 mA). To identify specific fractions after fixing nitrocellulose membrane treated with hyperimmune sera of rabbit peroxidase conjugate and substrate solution (benzidine HCI).

Specific antigenic polypeptide fraction obtained after purification of these components and having a molecular weight of 41.8 kDa, kongugiruut (sew) with the enzyme label (horseradish peroxidase) at the rate of 1 mg of enzyme per 5 mg of the polypeptide, using periodicly method (Enzyme-linked immunosorbent assay. Ed. GGG, Gmelinii. - M - 1988. - s-243).

Received protivopozarnyi antigen-enzyme conjugate (AG IFC) canned or subjected to freeze drying. A fundamental difference between the way of differentiation from post-infectious and post-vaccination heterologous (yersiniosis) antibodies is that the use of a highly specific and sensitive test system (ELISA) based on monospecific indicator component - labeled peroxidase isolated from a virulent strain of B. abortus 54 polypeptide fraction with mm 41,8 kDa, missing the vaccine (received 19.82, R-1096) strains of Brucella and heterologous microorganisms (Yersinia).

The method of differential diagnosis of brucellosis and method according to the teachings of the diagnostic preparation is carried out as follows.

The initial step in the manufacture of a diagnostic drug labeled with the enzyme horseradish peroxidase specific antigen Brucella, is the allocation of monospecific polypeptide antigenic fractions of a virulent strain of Brucella, which is absent in avirulent and vaccine strains of Brucella, which is the basic immunological indicator antigenic component of diagnosticum - protivopozharnogo enzyme conjugate (IFC).

Monospecific antigenic polypeptide fraction Brucella obtained by electrophoretic separation of lysates of several previously inactivated with gamma rays60With a dose of 3.0-3.5×104Gr.

For this purpose take virulent strains of species of B.abortus (54,544), B.suis (1330, S-40, 0302, 0303), B.canis (RM 6/66), B. melitensis (16M), Century obis (101, 31) and vaccine strains of Brucella (19, UV-1,82, R-1096), grow them on hepatic-peptone medium for 24 h, the resulting backass separated by centrifugation and diluted with saline to a concentration of 1·109M.K./cm3and subjected to radiation inactivation (radioactively) on gamma-install from the radiation source60With a dose of 3.0,is 3.5×104Gr.

Radioactiveman Brucella are lysed in a solution prepared by holding 0.062-0.125 M Tris HCl buffer, pH 6.8, containing 2-5% sodium dodecyl sulfate (SDS) and 5% β-mercap is aethanol. Bacterial cells lyse solution is heated on a boiling water bath for 1 min, then the samples poured the glycerol bromophenol blue to a final concentration of 10% and 0.001%, respectively. For holding the disk-electrophoresis of the samples in the wells of the concentration of the gel is to be applied in the amount of 50-100 μg protein. The mode of electrophoresis to the occurrence of the dye in the separating gel is 40 mA for 2 plates of the gel. Further, the electrophoresis is carried out at the force of the current of 80 mA for 2 plates of gel for 3-3,5 hours. Plates of the gel after electrophoresis is fixed in a solution containing 40% isopropyl alcohol and 10% acetic acid for 1 hours or leave overnight. Gels stained in 0.1%solution of Kumasi". Color fractionated in SDS page polypeptides silver carried out according to the method Eigenimage etc. /a Simple method for detection of proteins in polyacrylamide gel using impregnation their silver. - Ukrainian. biochem. log. - 1986 - T, No. 5. - Pp.62-65/ using formaldehyde solution. Densitometrically plates gels produced on the scanner Sharp 330 Yx in transmitted light. Determination of molecular mass and calculate the percentage content of protein (antigen) fractions exercise program Imayemachn 1 D prime.

When analyzing electrophoregram after the pickup of the lysates analyzed strains of Brucella in the gradient of 15-20% of the AAG in virulent strain 54 detects a wider range of polypeptides (up to 59 fractions) compared with all other strains and species of Brucella, have their number varies from 29 to 49 fractions with a molecular mass (mm) from 20 to 60 kDa. When comparative analysis of electrophoregram all tested species and strains of B. abortus 54 naturally find polypeptide fraction with a molecular weight of 41.8 kDa, which are absent in other species and strains of Brucella that has prinzipialnie is to use this component for constructing specific diagnosticum.

To determine the specificity and serological activity conducted by immunoblotting transfer antigenic fractions with plates page to nitrocellulose membrane ("Millipore") according to the method H.Touibm et al. // Proc. Natl. Acad. Sci. USA. - 1979. - V.76. - P.4350-4354) in buffer containing 0,024 M Tris, 0,M glycine and 20% menthol, using graphite plates (3 hours at 100 mA).

For the detection of serological activity and immunological competence fractions of Brucella after fixing nitrocellulose membrane processes relevant hyperimmune rabbit S-, SR-, R - sera to the antigens used in the experiments strains and species of Brucella.

Antigenic fraction showing the highest serological activity and specificity, i.e. the incoming immunological reaction only with antisera to virulent strains of the species B. abortus bovis isolated and used for further of the otopleniya antigenic protivopozharnogo enzyme conjugate (AG IFC).

For the preparation of AG IFC antigenic fraction from St, isolated as described above, is subjected to conjugation (merging) with the enzyme controllable periodic destruction method. As a label used horseradish peroxidase, and the labelling of the antigen is as follows.

Pre-horseradish peroxidase dissolved in distilled water to a final concentration of 5 mg/ml Concentration of enzyme in solution and the degree of purity of the enzyme spectrophotometric control, determining a measure of purity (Rz) and the concentration of enzyme in solution by the formula:

WithPHmg/ml: P=D430/D280

WithPHmg/ml=D403×0,44 times. sample

where D403, D280- the optical density of the solution, measured on the spectrophotometer at a wavelength of 403 nm and 280 nm, respectively;

0,44 - conversion.

To a solution of the enzyme was added 0.1 G. of aqueous solution of sodium metaperiodate (21,4 1 ml A.I) at a rate of 0.2 ml per 5 mg of peroxidase.

The mixture was kept at 18-24°20 min, shaking, and then cialiswhat against acetate buffer pH 4.4 in one day with three changes of buffer solution at a ratio equal to 1:500.

After dialysis, the enzyme solution is decanted from the dialysis bag, checking the pH, which should be in the range of 4-4,5, and stored at 0-4°when the ratio of the volume process is and enzyme buffer solution, equal to 1:500.

To the resulting solution was modified peroxidase add 0.2m carbonate-bicarbonate buffer pH 9,5-10,0 (1 ml of an enzyme solution add 0.1-0.15 ml buffer) to establish a pH in solution of the enzyme of 9.5 and 9.7, and then immediately added thereto and heated at 56°C for 30 min faction in the amount of not more than 1 ml, containing a protein that has 4 times the amount of peroxidase (i.e. for every 5 mg of peroxidase took 20 mg of fraction). The amount of antigenic fraction was adjusted to 1 ml of 0,01M carbonate-bicarbonate buffer pH of 9.5. The protein concentration is determined by the formula:

K=D280× and (dilution of sample)/1,3,

where 1,3 - constant coefficient calculation. The mixture was kept after checking the pH (which should be in the range of 9.5 to 10.0) at 18-24°C, With constant shaking for 2 hours, after which it cialiswhat against a 500-fold volume of 0.01 M phosphate-saline buffer solution pH of 7.2 to 7.4 at 4°during the day with a 2-3-fold change of the buffer.

Enzyme labeled antigen fraction - enzyme conjugate is preserved by adding thimerosal to a final concentration of 1:100,000 solution of the drug and stored in native form in the refrigerator.

Obtained as described above antigenic variant protivopozharnogo enzyme conjugate check on the activity and specificity is ity using the direct method enzyme-linked immunosorbent assay (ELISA). As a positive control, use a serum knowingly patients with brucellosis in animals, and as a negative control serum intact and vaccinated against bluetongue the animals, as well as heterologous control - serum media Brucella B. suis, B. canis, B. obis. Positive reaction with the serum of patients with brucellosis in animals with a maximum titer at a dilution of 1:100 and a negative reaction with sera of vaccinated protivopoloznymi vaccines and infectious heterologous antibodies animal indicates the specificity and activity of the obtained enzyme conjugates.

The formulation of differential diagnosis for brucellosis in cattle (cattle) as follows. The examined animals take blood from the jugular vein in a volume of 10 cm3and get a serum standard method.

The resulting serum is used as antibodies in the live version of ELISA, formulation and evaluation of results is carried out in accordance with the methodological recommendations Hramina and Gholizade ("Immunological methods", M., 1987, s.162-170).

As a specific antigen in a live version of the ELISA used the above described method antigenic variant enzyme conjugate (AG IFC).

The reaction is accompanied by the trail of the sponding controls:

serum intact (healthy, unvaccinated animals);

serum inoculated St animals;

serum inoculated .R-1096 animals;

- serum media Y. enterocolitica;

serum contaminated STV suis;

serum contaminated STV bovis;

serum contaminated STV melitensis.

For a positive reaction in ELISA accept a dilution of 1:100 factor specificity (PSC) - 2.1 and higher negative reactions with heterologous and whole group sera from vaccinated brucellosis vaccines, and other Brucella species).

The positive reaction of the ELISA used serum in dilutions 1:100 and higher (factor specificity PSC - 2.1 and above) indicates the presence in the body of the subject animals virulent strain of Brucella species B. abortus.

The method of differential diagnosis of brucellosis tested in the laboratory (Guinea pigs and rabbits) and by a Commission on agriculture (beef cattle) animal affected with brucellosis farms.

The method of differential diagnosis of brucellosis and the method of producing drug for its implementation are illustrated by the following examples.

Example 1. Obtaining specific antigenic fractions of B. abortus.

Brucella antigen was obtained by standard methods by termextraction Brucella, literaturnogo destruction, gamma-irradiation, ethanol and phenol extraction, lizirovania cells with subsequent gel electrophoresis in polyacryamide gel (SDS page). Spectrometric analysis of the antigens showed that they contain no more than 2-3 of the main fractions of antigens that have entered the serological reactions with S-, SR-, and R - sera obtained from the native species of Brucella C. abortus, B. suis, bovis, canis, melitensis, and from immunized protivopoloznymi animal vaccines. Use for this purpose methods of radioactively, destruction of cells in the lytic solution, followed by gel-electrophoresis in SDS page provides selection strictly specific component B. abortus 54, which reacts ELISA only with antisera contaminated virulent strains of the species B. bovis.

Example 2. Obtaining antigen-enzyme conjugate.

Enzyme conjugate was obtained by blending antigenic fractions of Brucella St with enzyme-labeled enzyme horseradish peroxidase in the ratio of antigen (protein) and labels 1:5, 1:10, 1:15, 1:20, 1:25 when the modes of conjugation, 5, 10, 15, 20, 25 minutes

It was found that the optimal time of conjugation was 20 minutes, and the ratio of antigen and label - 1:20.

Changing modes of conjugation (component ratio, time of conjugation, stitching, metal and other parameters) did not provide a receipt conjugates with sufficient serological activity for the titer of the antigen does not exceed 1:30.

Example 3. Effective differentiation of post-infectious antibodies from vaccination was tested on Guinea pigs. To this end 40 Guinea pigs were subjected to infection with virulent strains of the causative agent of brucellosis B. abortus 54 (1), 99 (2), 544-(3-I) group (5 animals per strain)immunized with vaccine strains 19 (4), 82 (5-I) and R-1096 (6th) (5 Guinea pigs to each variant of the vaccine)and also infected by the pathogen Yersinia (Y. enterocolitica) (7); 8 group animals were immunized and not infected (biological control). All animals in the dynamics (3, 7, 14, 21 and 28 days after injection of Brucella antigens) took blood samples for serological studies in ELISA. It is established that during all periods of the study in control and immunized animals (4-I, 5-I, 6-I, 7-I and control group) antibodies were not detected. Unlike immunized got infected with virulent strains of the causative agent of brucellosis (1-I, 2-I, 3-I group) were found protivopozarnyi antibodies at titers of 1:4-1:8, 1:16-1:32, 1:64-1:128 7, 14, 21 and 28 days after infection, respectively. In a parallel study of the tested sera in PA, PCK and RIGA in all immunized and infected with virulent strains of Brucella were detected antibodies in titers of 1:10-1:50 on 14, 21 and 28 days after infection the immunization. Neither the title nor the appearance of antibodies to differentiate their class, i.e. post-vaccination or postinfection, was not possible.

Example 4. The effectiveness of the proposed method for differential diagnosis of brucellosis and differentiating patients with brucellosis the animals from vaccinated tested in cattle in a safe, inoculated strains 19 and 82, as well as in not safe for brucellosis of cattle farms of the Tula region and the Republic of Tatarstan. Has determined that the regulated when brucellosis test systems (RA, PCK, BPO) using well-known diagnosticum (single Brucella antigen) did not allow to differentiate patients with brucellosis the animals from vaccinated because serum as vaccinated and spontaneously infected animals reacted positively with a known antigen. Unlike conventional test systems offer the test system (ELISA) using the proposed diagnosticum (enzyme conjugate on the basis of polypeptide fractions of Brucella from St) allowed to identify the serum of patients with brucellosis from vaccinated against this disease of animals.

1. The method of differential diagnosis of patients with brucellosis the animals from vaccinated, characterized in that conduct sør the logical analysis of blood sera of animals in the enzyme-linked immunosorbent assay (ELISA) using antigenic protivopozharnogo enzyme conjugate, representing labeled with horseradish peroxidase electrophoretic purified polyacrylamide gel antigenic polypeptide fraction virulent strain of C. abortus 54 with a molecular weight of 41.8 kDa, and differentiate patients with brucellosis the animals from vaccinated when detected in the sera of infected animals protivopolozhnyh antibodies in dilutions of 1/100 or higher, the degree of intensity of the reaction XP,1 and above and in the absence of protivopolozhnyh antibodies in sera from vaccinated.

2. The method of obtaining protivopozharnogo antigen-enzyme conjugate (IFC) for the differential diagnosis of patients with brucellosis the animals from vaccinated, characterized in that the virulent strain of Brucella species C. abortus 54 inactivating gamma-rays60With a dose of 3.0-3.5×104Gr, are lysed in a solution prepared by holding 0.062-0.125 M Tris HCL buffer, pH 6.8, containing 2-5% sodium dodecyl sulfate and 5% β-mercaptoethanol, put the samples into the wells concentrating gel in the amount of 50-100 μg protein, conduct electrophoresis with an electric current of 40 mA on the two plates of the gel prior to the occurrence of the dye in the gel and then when the power current of 80 mA on two plates 3-3,5 h, fixed plate in a solution containing 40% isopropyl alcohol and 10% acetic acid for 1 h, stained 0.1%solution of Kumasi" diamond goal is nd R-250, then the fraction of polypeptides paint silver with the use of a solution of formaldehyde, densitometric plate of the gel on the scanner SHARP-330Y x in transmitted light, determine the molecular weight and immunological competence fractions method immunobloting, antigenic polypeptide fraction virulent strain of Brucella C. abortus 54 with a molecular weight of 41.8 kDa kongugiruut with the enzyme label is horseradish peroxidase at a rate of 1 mg protein per 20 mg of peroxidase with constant stirring at room temperature for 20 minutes



 

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