RU2300107C2 - Method for differential diagnostics of bovine brucellosis and a method of preparing a preparation for implementation thereof - Google Patents

Abstract

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

Description

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

Several methods for serological and differential diagnosis of bovine brucellosis are known. These are such as agglutination reaction (RA), complement fixation reaction (RAC), long-term complement fixation reaction (RDSK), indirect hemagglutination reaction (RNGA), rose bengal test (RBP) and others.

The disadvantage of these methods is the lack of strict specificity of these test systems, since in many cases in animals reactions that are not associated with the development of an infectious brucellosis process are detected. This is due to the fact that as a main immunological component in these test systems, a single brucellosis antigen is used that reacts with both S- and RS-antibodies present both in patients with brucellosis and inoculated with anti-brucellosis vaccines, as well as in animals - carriers of Yersinia - Y.enterocolitica.

The closest technical solution, selected as a prototype, is ELISA (enzyme-linked immunosorbent assay), in which for the differential diagnosis of brucellosis, serological indication of brucella is performed in veterinary surveillance facilities using monoclonal antibodies (MCAs). At the same time, monoclonal antibodies are added to the tablets sensitized with brucellosis antigen, and after exposure at a temperature of 37-38 ° C for 1 hour, an antispecies enzyme-linked immunosorbent conjugate is added. The presence of antigens in the test sample is judged by the degree of color intensity of the contents of the wells. The reaction is considered positive when the color intensity of 2-4 cross in dilutions 1 / 16-1 / 32. (Plotnikova E.M., Salmakov K.M. - Development of methods and means of immunomonitoring of animal brucellosis. - Veterinary medicine. - 2003. - No. 12. - P.16-18.)

However, differential diagnosis of brucellosis using the ELISA test by indicating the causative agent of brucellosis (brucellosis antigen) is fraught with technological difficulties, because the process of indicating the pathogen is a very time-consuming and difficult task, since it is necessary to prepare samples for investigation, accumulate bacterial biomass, and process antigen IFA material and production.

The disadvantage of this method is the complexity and high cost of obtaining MCA for ELISA, long and multi-stage preparation for the study, the accumulation of bacterial biomass, processing of antigen-containing material, etc. In this case, the desired agent may not be isolated, since the result of preliminary bacteriological indication with a small number of microbes in the studied samples (less than 10 ⁴ microbial cells per 1 g (ml) of sample) may be negative, which reduces the sensitivity and effectiveness of serological diagnosis of brucellosis. With this method, the main goal is not achieved - the differentiation of virulent strains of the pathogen from avirulent and vaccine variants of brucella, which is associated with the use of the R-form antigen of brucella for hyperimmunization of animal donors of specific antibodies.

The objective of the invention is to develop a method that increases the specificity of the ELISA test and allows the differentiation of post-vaccination immunological reactions from post-infectious, as well as the differentiation of cross-reactions induced by microorganisms having antigenic affinity with brucella, especially Yersinia entericolica.

The problem is solved through the use of serological analysis of blood serum of the studied animals in an enzyme-linked immunosorbent assay (ELISA), which is carried out using an antigenic variant of the enzyme-linked immunosorbent enzyme (IFC AG), in which the polypeptide fraction of the B. vortus 54 virulent strain with molecular weighing 41.8 kDa, which is absent in vaccine (B. abortus 19.82, R-1096) and other virulent species (B. suis, B. obis) brucella, and the presence of of specific post-infectious antibodies in the studied sera, according to the degree of reaction intensity (CSP) and the titer of post-infectious antibodies in ELISA, they make a differential diagnosis for brucellosis, for the diagnostic titer of post-infectious antibodies they take a positive reaction in dilutions of the studied sera 1: 100 and higher with a specificity coefficient (Csp) -2.1 and higher.

The invention also lies in the fact that the method for producing an antigenic variant of an enzyme-linked immunosorbent conjugate (IHF antigens) for differentiating post-infectious antibodies from post-vaccination antibodies involves the radioactivation of brucella and their subsequent lysis, isolation of a specific antigenic fraction of the virulent B. abortus 54 strain by electrophoretic method in polyacrylamide in a concentration gradient of 10-20% in the presence of Na dodecyl sulfate (SDS), current strength of 80 mA and exposure time of 2.5-3.0 hours, fixation of gel plates in a solution containing 40% of propyl alcohol and 10% acetic acid, followed by staining the gel in 0.1% Coomassie brilliant blue R-250, additional staining of the polypeptide fractions with silver using a formaldehyde solution, determination of molecular weights of fractionated polypeptides using a calibration graph based on standard markers : BSA (67 kDa), EA (43 kDa) and cytochrome C (12.3 kDa), transfer of antigenic fractions from PAAG plates to the nitrocellulose membrane ("Miliipore") with a buffer containing 0.024 M Tris, 0.076 M glycine and 20 % methanol using graphite plates (3 hours at 100 mA). To identify a specific fraction after fixation, the nitrocellulose membrane is treated with hyperimmune rabbit sera with a peroxidase conjugate and a substrate solution (benzidine HCI).

A specific polypeptide antigenic fraction obtained after purification with the indicated components and having a molecular weight of 41.8 kDa is conjugated (crosslinked) with the enzyme label (horseradish peroxidase) at the rate of 1 mg of the enzyme per 5 mg of the polypeptide using the periodate method (Enzyme Immunoassay./ Ed. G.G. Ngo, G.M. Lenhoff. - M. - 1988. - p. 240-243).

The obtained anti-brucellosis antigenic enzyme-linked immunosorbent conjugate (AG IFC) is preserved or freeze dried. A significant difference in the method of differentiating post-infectious from post-vaccine and heterologous (yersiniosis) antibodies is that for this purpose they use a highly specific sensitive test system (ELISA) based on a monospecific indicator component - a peroxidase-labeled polypeptide fraction isolated from virulent B. abortus 54 strain with mm. 41.8 kDa, which is absent in vaccine (19.82, R-1096) strains of brucella and heterologous microorganisms (Yersinia).

The method of differential diagnosis of brucellosis and the method of obtaining a diagnostic drug is as follows.

The initial step in the manufacture of a diagnostic drug - horseradish peroxidase-labeled specific antigen of brucella, is the isolation of a monospecific polypeptide antigenic fraction of the virulent strain of brucella, which is absent in avirulent and vaccine strains of brucella, which serves as the main immunological indicator component of the antigen diagnostic antigen-conjugation-antigens.

The monospecific polypeptide antigenic fraction of brucella is obtained by electrophoretic separation of brucella lysates previously inactivated with ⁶⁰ Co gamma rays at a dose of 3.0-3.5 × 10 ⁴ Gy.

For this purpose, virulent strains of the species B. abortus (54,544), B.suis (1330, S-40, 0302, 0303), B.canis (RM 6/66), B. melitensis (16M), B. obis ( 101, 31) and vaccine strains of brucella (19, UV-1.82, R-1096), they are grown on a liver-peptone medium for 24 hours, the obtained bacmass is separated by centrifugation and diluted with physiological saline to a concentration of 1 · 10 ⁹ m. K. / cm ³ and subjected to radiation inactivation (radioactivation) on a gamma installation with a radiation source of ⁶⁰ Co in a dose of 3.0, -3.5 × 10 ⁴ Gy.

Radioactivated brucellas are lysed in a solution prepared on 0.062-0.125 M Tris HCl buffer, pH 6.8, and containing 2-5% sodium dodecyl sulfate (SDS) and 5% β-mercaptoethanol. Bacterial cells in a lysing solution are heated in a boiling water bath for 1 min, then glycerol with bromphenol blue is added to the samples to a final concentration of 10% and 0.001%, respectively. To conduct disk electrophoresis, samples in the wells of a concentrating gel are applied in an amount of 50-100 μg per protein. The electrophoresis mode before the dye enters the separable gel is 40 mA per 2 gel plates. Further electrophoresis is carried out at a current strength of 80 mA per 2 gel plates for 3-3.5 hours. After electrophoresis, the gel plates are fixed in a solution containing 40% isopropyl alcohol and 10% acetic acid for 1 hour or left overnight. The gels are stained in a 0.1% Coomassie solution. The polypeptides fractionated in PAG are stained with silver according to the method of E.I. Grebinozhko et al. / A simple method for detecting proteins in a polyacrylamide gel by impregnating them with silver. - Ukrainian. biochem. Journal. - 1986 - T. 58, No. 5. - S.62-65 / using a solution of formaldehyde. Densitotometry of the gel plates is carried out on a Sharp 330 Yx scanner in transmitted light. The determination of molecular weights and the calculation of the percentage of protein (antigenic) fractions is carried out according to the Imayemachn 1 D prime program.

When analyzing electrophoregrams after distillation of the lysates of the studied brucella strains in a 15–20% PAAG gradient, virulent strain 54 reveals a wider range of polypeptides (up to 59 fractions) compared to all other brucella strains and species, in which their number varies from 29 to 49 fractions with a molecular weight (m.m.) of 20 to 60 kDa. Comparative analysis of electrophoregrams of all tested species and strains in B. abortus 54 naturally reveals a polypeptide fraction with a molecular weight of 41.8 kDa, which is absent in other species and strains of brucella, which is of fundamental importance for using this component to construct a specific diagnosticum.

In order to determine the specificity and serological activity, immunoblotting is carried out by transferring antigenic fractions from PAAG plates to a nitrocellulose membrane (Millipore) according to the method of H. Touibm et al. // Proc. Natl. Acad. Sci. USA - 1979. - V.76. - P.4350-4354) in a buffer containing 0.024 M Tris, 0.076 M glycine and 20% menthol, using graphite plates (3 hours at 100 mA).

To identify the serological activity and immunological competence of the brucella fractions after fixation, the nitrocellulose membrane is treated with the corresponding hyperimmune rabbit S-, SR-, R-serums to the antigens of the brucella strains and species used in the experiments.

The antigenic fraction exhibiting the highest serological activity and specificity, i.e. entering into an immunological reaction only with antisera to virulent strains of the species B. abortus bovis, is isolated and used in the future for the manufacture of antigenic anti-brucellosis enzyme-linked immunosorbent conjugate (AG IFC).

For the preparation of AG IFC, the antigenic fraction from 54, isolated by the above method, is subjected to conjugation (crosslinking) with the enzyme by the periodic method. Horseradish peroxidase is used as a label, and antigen labeling is carried out as follows.

Previously, horseradish peroxidase is dissolved in distilled water to a final concentration of 5 mg / ml. The concentration of the enzyme in the solution and the purity of the enzyme are controlled spectrophotometrically, determining the purity index (Rz) and the concentration of the enzyme in the solution according to the formula:

With _HRP mg / ml: R. = D ₄₃₀ / D ₂₈₀

With _HRP mg / ml = D ₄₀₃ × 0.44 div. sample

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

0.44 - conversion factor.

0.1 N was added to the enzyme solution. an aqueous solution of sodium metaperiodate (21.4-1 ml ai) at the rate of 0.2 ml for every 5 mg peroxidase.

The mixture is kept at 18-24 ° C for 20 minutes, shaking, and then dialyzed against an acetate buffer, pH 4.4 for a day with a three-time change of the buffer solution at a ratio of 1: 500.

At the end of 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 ° C with a ratio of the volume of the solution of the enzyme buffer solution equal to 1: 500.

0.2M carbonate-bicarbonate buffer pH 9.5-10.0 is added to the resulting modified peroxidase solution (0.1-0.15 ml buffer is added to 1 ml of the enzyme solution) until the pH in the enzyme solution is 9.5-9, 7, after which fractions immediately heated up to 56 ° C for 30 min are added to it in a volume of not more than 1 ml containing an amount of protein 4 times the amount of peroxidase (i.e., 20 mg fractions were taken for every 5 mg of peroxidase). The volume of the antigenic fraction is adjusted to 1 ml with 0.01 M carbonate-bicarbonate buffer pH 9.5. The protein concentration is determined by the formula:

K = D ₂₈₀ × a (sample dilution) / 1.3,

where 1.3 is a constant calculation coefficient. The mixture is maintained after checking the pH (which should be in the range of 9.5-10.0) at 18-24 ° C, with constant shaking for 2 hours, after which it is dialyzed against a 500-fold volume of 0.01 M phosphate-salt buffer solution pH 7.2-7.4 at 4 ° C for a day with a 2-3-fold change of buffer.

An antigenic fraction labeled with an enzyme — an enzyme-linked immunosorbent conjugate — is preserved by the addition of thiolate to a final concentration of 1: 100000 in a solution of the drug and stored in its native form under refrigerated conditions.

Obtained by the above method, the antigenic variant of anti-brucellosis enzyme-linked immunosorbent conjugate is checked for activity and specificity using the direct enzyme-linked immunosorbent assay (ELISA). In this case, the sera of animals known to be ill with brucellosis are used as a positive control, and the sera of intact and vaccinated animals against this disease are used as a negative control, and the brucella carriers of B. suis, B. canis, B. obis are used as a heterologous control. A positive reaction with the serum of animals with brucellosis in animals with a maximum titer of 1: 100 dilution and a negative reaction with the sera of vaccinated anti-brucellosis vaccines and animals contaminated with heterologous antibodies indicates the specificity and activity of the obtained enzyme immunoassay conjugates.

The differential diagnosis for bovine brucellosis (cattle) is as follows. The examined animals take blood from the jugular vein in a volume of 10 cm ³ and obtain serum by the conventional method.

The obtained serum is used as antibodies in the direct version of ELISA, the formulation and evaluation of the results of which is carried out in accordance with the methodological recommendations of H. Fremel and G. Holtsheidt ("Immunological methods", M., 1987, p.162-170).

As a specific antigen in the direct variant of ELISA, the antigenic version of the enzyme-linked immunosorbent conjugate (AG IFC) obtained by the above method is used.

The reaction is accompanied by the following controls:

- serum of intact (healthy, unvaccinated animals);

- serum of vaccinated 19 animals;

- serum of vaccinated R-1096 animals;

- serum of carriers of Y. enterocolitica;

- serum contaminated pcs. B. suis;

- serum contaminated pcs. B. bovis;

- serum contaminated pcs. B. melitensis.

For a positive ELISA, a dilution of 1: 100 with a specificity coefficient (Ksp) of 2.1 and higher is taken with a negative reaction with heterologous and group-wide sera (from vaccinated with brucellosis vaccines and other types of brucella).

A positive reaction of ELISA with used sera at dilutions of 1: 100 and higher (Ksp specificity coefficient is 2.1 and higher) indicates the presence of a virulent strain of B. abortus brucella strain in the examined animals.

The method of differential diagnosis of brucellosis has been tested on laboratory (guinea pigs and rabbits) and on a commission on agricultural (cattle) animals in brucellosis dysfunctional farms.

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

Example 1. Obtaining a specific antigenic fraction of B. abortus.

Brucellosis antigen was obtained by conventional methods by thermoextraction of brucella, ultrasonic destruction, gamma irradiation, ethanol and phenol extraction, cell lysis, followed by polyacrimide gel electrophoresis (PAAG). Spectrometric analysis of the obtained antigens showed that they contain no more than 2-3 main fractions of antigens that entered into serological reactions with both S-, SR-, and R-sera obtained from carriers of brucella species B. abortus, B. suis, In bovis, In canis, In melitensis, as well as from animals immunized with brucellosis vaccines. Using for this purpose methods of radioactivation, destruction of cells in a lysing solution followed by gel electrophoresis in PAGE ensures the isolation of a strictly specific component of B. abortus 54, which reacted with ELISA only with antisera of contaminated virulent strains of B. bovis species.

Example 2. Obtaining antigenic enzyme-linked immunosorbent conjugate.

An enzyme-linked immunosorbent conjugate was obtained by cross-linking the piece 54 brucell antigenic fraction with the enzyme-linked immunosorbent label-enzyme horseradish peroxidase in the ratio of antigen (protein) and label 1: 5, 1:10, 1:15, 1:20, 1:25 with conjugation modes 5, 10, 15, 20, 25 minutes

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

Changes in conjugation modes (component ratios, conjugation time, crosslinking, labels, and other parameters) did not provide conjugates with sufficient serological activity, because the antigen titer did not exceed 1:30.

Example 3. The effectiveness of the method of differentiation of post-infectious antibodies from post-vaccination antibodies was tested in guinea pigs. To this end, 40 guinea pigs were infected with virulent strains of the brucellosis pathogen B. abortus 54 (1st), 99 (2nd), 544- (3rd) group (5 animals per strain), immunized with vaccine strains 19 (4th), 82 (5th) and R-1096 (6th) (5 guinea pigs for each variant of the vaccine), and also were infected with the causative agent of yersiniosis (Y. enterocolitica) (7th group); The 8th group of animals was not immunized or infected (biological control). Blood samples were taken from all animals in dynamics (3, 7, 14, 21, and 28 days after the administration of Brucella antigens) for serological testing in ELISA. It was found that in all periods of the study, antibodies were not detected in the control and immunized animals (4th, 5th, 6th, 7th and control groups). In contrast to the brucellosis pathogen immunized in infected with virulent strains (1st, 2nd, 3rd groups), anti-brucellosis antibodies were detected in titers from 1: 4-1: 8, 1: 16-1: 32, 1:64 -1: 128 on 7, 14, 21 and 28 days after infection, respectively. A parallel study of the tested sera in PA, PCK and RIGA for all immunized and infected with virulent strains of brucella revealed antibodies in titers of 1: 10-1: 50 on days 14, 21 and 28 after infection and immunization. At the same time, neither by titer nor by the timing of the appearance of antibodies to differentiate them by class, i.e. post-vaccination or post-infectious, was not possible.

Example 4. The effectiveness of the proposed method for the differential diagnosis of brucellosis and the possibility of differentiating animals with brucellosis from vaccinated animals were tested on cattle in safe, vaccinated with strains 19 and 82, as well as in unsuccessful cattle brucellosis farms in the Tula region and the Republic of Tatarstan. It was established that the results of test systems regulated by brucellosis (RA, PCK, RBP) using the well-known diagnosticum (a single brucellosis antigen) did not allow differentiating animals with brucellosis from vaccinated animals, since the sera of both vaccinated and spontaneously infected animals reacted positively with the known antigen . In contrast to the generally accepted test systems, the proposed test system (ELISA) using the proposed diagnosticum (enzyme-linked immunosorbent conjugate based on the polypeptide fraction of brucella from pcs. 54) made it possible to identify the sera of patients with brucellosis from animals vaccinated against this disease.

Claims (2)

1. A method for the differential diagnosis of patients with brucellosis of animals from vaccinated, characterized in that they carry out serological analysis of animal blood serum in an enzyme-linked immunosorbent assay (ELISA) using an antigenic anti-brucellosis enzyme-linked immunosorbent conjugate, which is a horseradish peroxidase-labeled electrophoretically purified polyacrylamide antigen polypeptide abortus 54 with a molecular weight of 41.8 kDa, and differentiate animals with brucellosis from vaccines th e detection in sera from patients animals antibrucellar antibodies at dilutions of 1/100 or more, the degree of reaction intensity Ksp.2,1 and above in the absence antibrucellar antibodies in sera from vaccinated. 2. A method of obtaining anti-brucellosis antigenic enzyme-linked immunosorbent conjugate (IFC) for the differential diagnosis of patients with brucellosis animals from vaccinated, characterized in that the virulent strain of brucella species B. abortus 54 inactivate ⁶⁰ Co gamma rays at a dose of 3.0-3.5 × 10 ⁴ Gy, lysed in a solution prepared on 0.062-0.125 M Tris HCL buffer, pH 6.8, containing 2-5% sodium dodecyl sulfate and 5% β-mercaptoethanol, apply samples to wells of a concentration gel in an amount of 50-100 μg per protein conduct electrophoresis at a current of 40 mA on two faces gel before entering the dye into the gel and then at a current strength of 80 mA on two plates for 3-3.5 hours, fix the plates in a solution containing 40% isopropyl alcohol and 10% acetic acid for 1 hour, stain in 0.1% -th solution of Coomassie brilliant blue R-250, then the polypeptide fractions are stained with silver using a formaldehyde solution, densitometry of the gel plates on a SHARP-330Y x scanner in transmitted light, the molecular weight and immunological competence of the fractions are determined by immunoblotting, the polypeptide antigenic fraction a virulent B. abortus 54 brucella strain with a molecular weight of 41.8 kDa is conjugated to the enzyme label horseradish peroxidase at the rate of 1 mg protein per 20 mg peroxidase with constant stirring at room temperature for 20 minutes.