RU2796393C1 - METHOD FOR RAPID QUANTITATIVE DETERMINATION OF THE LEVEL OF HUMORAL IMMUNITY OF ANIMALS TO STRAIN O N2311/ZABAIKALSKY/2016 OF GENOTYPE O/ME-SA/Ind-2001 OF FOOT AND MOUTH DISEASE VIRUS AFTER VACCINATION USING LIQUID-PHASE "SANDWICH" ELISA - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the development of a method for rapid quantitative determination of the level of humoral immunity of animals to strain O No. 2311/Zabaikalsky/2016 of the genotype O/ME-SA/Ind-2001 of the foot-and-mouth disease virus after vaccination using a liquid-phase "sandwich" ELISA variant. The proposed method involves the use of immunospecific and non-specific components, allows to determine the percentage of inhibition and quantify the level of humoral immunity of animals against strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001 after vaccination. The developed method is characterized by high diagnostic sensitivity, specificity and accuracy for the quantitative determination of antibody titer against FMDV strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001.

EFFECT: invention provides obtaining a result within 4 hours with high reliability.

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Description

The invention relates to the field of veterinary virology and biotechnology, in particular, the development of a method for rapid quantitative determination of the level of humoral immunity of animals to strain O No. 2311/Zabaikalsky/2016 of the genotype O/ME-SA/Ind-2001 of foot-and-mouth disease virus after vaccination using a liquid-phase blocking indirect " sandwich" ELISA variant.

Foot-and-mouth disease is a particularly dangerous, quarantine, highly contagious disease of artiodactyl and corn-footed animals, which is caused by an RNA (+)-containing virus of the Picornavirales order of the Picornaviridae family of the Aphthovirus genus with a nucleic acid length of about 8500 bp. [1]. When the FMD virus reproduces in biological systems, a 146S component (complete particles, the main immunogenic component) is formed, consisting of one viral RNA molecule and 60 copies of the polypeptide, each of which is represented by a complex of proteins VP ₁ -VP ₂ -VP ₃ -VP ₄ [2] .

There are 7 FMD serotypes, namely O, A, C, Asia-1, SAT-1, SAT-2 and SAT-3. Each serotype is genetically divided into different topotypes, genetic lines and sub-lines. Differences between them are determined by analyzing the nucleotide sequence of the 1D gene (VP ₁ protein), which is characterized by high genomic and antigenic variability [3].

Within the seven serotypes, more than 60 genetic lines and sublines have been described. It is believed that antibodies against one serotype do not provide immune protection against another serotype, and even more so some other genetic lines. However, FMD viruses can show partial cross-protection within the same serotype [4].

The observed genetic variation in the FMDV genome is the result of two processes. First, these are errors that result from viral RNA replication and the lack of repair of the encoded RNA-dependent RNA polymerase. Secondly, competitive selection, which affects the genome. In the natural environment, as a result, isolates of new genetic lines that include in their genome the characteristics required for this environment will prevail [5].

In RNA viruses, genome variations are favored by high mutation rates during virus replication, and emerging lineages and sublines are due to partial nucleotide substitutions and recombination. Recombination is the exchange of genetic material between two non-segmented RNA genomes. It occurs between viruses of the same serotype [6]. Recombinations in the nucleic acid of FMDV occur more easily in the 3'-part of the genome than in regions encoding structural viral proteins. Mutations as a result of recombination can lead to the exchange of genetic material and the formation of new antigenic variants, which often lead to changes in cell tropism [4]. One or more amino acid substitutions on the surface of viral particles can lead to altered receptor recognition and utilization. As a result, the same virus can acquire the ability to penetrate cells using alternative receptors [1, 2].

Antigenic variations arise from amino acid mutations that alter the recognition of viral proteins by the body's immune system. The superficially open structures of the virus are particularly susceptible to immune attack. The process of emergence of new antigenic forms is determined by the complex interaction of viral factors and surface proteins of host cells. Mutations in the genes encoding capsid proteins can lead to antigenic changes and the emergence of new genetic lines [1, 2].

The most variable of all FMDV proteins is VP ₁ because it accounts for about 90% of mutations in all structural genes. The most variable regions are regions 40–60, 130–160, and 190–213 a.a. [1]. The site of the surface protein VP ₁ in the region of 130-160 a.a. is characterized by high variability, since it is involved in the process of binding to host cell receptors [4]. The variability of this region enables FMDV to interact with receptors on different cell types and facilitates the transition from one host species to another. FMD virus serotypes are on average 86% identical in sequence, although VP ₁ is significantly more variable. Nucleotide sequences of the VP ₁ coding region are used to genetically characterize FMD strains because of their importance for viral attachment and cell entry, protective immunity, and serotype specificity.

The GenBank database contains nucleotide sequences for various genetic lines and sublines [7]. In this analysis, we are interested in the FMD virus serotype O, which is the most studied and widespread throughout the world. It is divided into 11 following topotypes: EAST AFRICA 1-4 (East Africa) (EA-1-4), SOUTHEAST ASIA (Southeast Asia) (SEA), EUROPE-SOUTH AMERICA (Europe-South America) (EURO-SA ), INDONESIA-1 and 2 (Indonesia-1 and 2) (ISA-1 and -2), CATHAY (China), MIDDLE EAST-SOUTH ASIA (ME-SA) (Middle East-South Asia) and WEST AFRICA (Western Africa) (WA) [4].

For the manufacture of vaccines and diagnostic kits for determining the level of humoral immunity, various strains of type O of the foot-and-mouth disease virus have been used for more than 65 years [8]. In 1957, strain O No. 194 / Volgogradsky / 67 (genotype O / EURO-SA) was isolated in the Volgograd region of the Russian Federation, in 1966 the strain O / Chechen-Ingush / 66 (genotype O / EURO-SA) was obtained, which were actively used for the manufacture of inactivated culture vaccines and the development of methods for determining the level of humoral immunity.

In April 2000, from pigs in the OPH "Stepnoye" with. Elite Ussuriysky district of Primorsky Krai isolated strain O/Primorsky/2000 (genotype O/ME-SA/PanAsia) of foot-and-mouth disease virus. Known strains O/Taiwan/97 and O No. 2222/Taiwan/2012 of FMD virus (genotype O/CATHAY/Cam 94). In 2012, strain O no. 2147/Primorsky/2012 (genotype O/ME-SA/PanAsia) was isolated from sick cattle in the village of Usachovka, Khorolsky district, Primorsky Krai. In May 2014, in the village of Prokhory, Spassky District, Primorsky Territory, in the buffer zone of the Russian Federation, an outbreak of FMD caused by FMDV genotype O/SEA/Mya-98 strain O No. 2212/Primorsky/14 was registered.

Between 2015 and 2016 on the territory of Saudi Arabia, the United Arab Emirates, Nepal, Iran, Thailand, Armenia, Turkey, the FMDV of the O/ME-SA/Ind-2001 genotype was detected for the first time. FMD outbreaks of this genotype have been recorded in the Russian Federation since 2016. FMDV of this genotype was isolated from the buffer zone of the Russian Federation in November 2016, the strain of which was named O No. 2311/Zabaikalsky/2016 [9]. On the territory of the Russian Federation, this strain was isolated for the first time. Currently, all of the above strains are stored in the State Collection of Microorganism Strains of the Federal State Budgetary Institution "Federal Center for Animal Health" (FGBU "ARRIAH").

According to the results of a comparative analysis of nucleotide and amino acid sequences, the O/ME-SA/Ind-2001 genotype strain differs significantly from previously used vaccine strains of other genotypes. Vaccines obtained using the antigen of the strains listed above do not allow to achieve a protective level of humoral immunity against strain O No. 2311/Zabaikalsky/2016 of the foot-and-mouth disease virus.

Immunization is an effective tool to fight the disease. For the manufacture of vaccines against foot-and-mouth disease type O, production strains of foot-and-mouth disease virus are used. In connection with the emergence of a representative of a new genotype of the FMD virus and the need to protect animals from it, the FGBI “ARRIAH” developed a vaccine that includes the antigen of the strain O/Zabaikalsky/16 of the FMD virus [10].

There was a need to create a method for quickly determining the level of humoral immunity of animals to strain O No. 2311/Zabaikalsky/2016 of the foot-and-mouth disease virus (genotype O/ME-SA/Ind-2001) using a liquid-phase "sandwich" ELISA variant.

The level of humoral immunity of the vaccinated livestock is determined by detecting antibodies specific to the structural proteins of the FMDV VP ₁ , VP ₂ , VP ₃ , VP ₄ . In accordance with the recommendations of the OIE Guidelines (OIE) [6], neutralization reaction and enzyme immunoassay (ELISA) are used to determine the immune status of animals. The neutralization reaction has a number of limitations, in particular, the long reaction time (at least 3 days), the high cost of the procedure associated with the use of a cell line, the presence of a CO ₂ incubator, and also involves the use of a non-inactivated foot-and-mouth disease virus, which is a biological agent II pathogenic groups. At the same time, ELISA has many advantages, including rapidity, high specificity and sensitivity, suitability for large-scale screening of field samples, and no requirements for special laboratory conditions, such as cell culture or carbon dioxide environment. Based on this, the ELISA method should be used to determine the level of humoral immunity in vaccinated animals.

Currently, there are direct and indirect variants of ELISA. The advantages of the direct ELISA variant are the speed of analysis, since only one IgG variant is used and cross-reactivity of secondary antibodies is eliminated. At the same time, there are some disadvantages, namely, a decrease in the immune activity of primary antibodies due to the negative influence of enzymes or labels, the lack of flexibility in choosing a primary antibody label depending on the task of the study, as well as minimal signal enhancement.

The indirect ELISA has significant advantages over the direct ELISA, namely: 1) the presence of a wide variety of labeled secondary antibodies, 2) versatility (many primary antibodies can be obtained from one species, and the same labeled secondary antibody can be used to detect ), 3) the maximum immune activity of the main antibody is preserved, since it is not labeled, 4) increased sensitivity, because each primary antibody contains several epitopes that can be bound by a labeled secondary antibody, which makes it possible to enhance the detected signal [11, 12].

Currently widely used "sandwich"-ELISA, which usually requires the use of pairs of matched antibodies, where each antibody is specific for a different, non-overlapping epitope of the antigen. The first antibodies are called sensitizing and are designed to capture the antigen. The second antibodies - detector, are necessary for the detection of the antigen. The advantages of the "sandwich" ELISA are: 1) high specificity; 2) suitable for crude animal serum samples; 3) high reaction sensitivity; 4) the possibility of quantitative analysis.

ELISA is also divided into solid-phase and liquid-phase variants. Currently, the solid phase variant is widely used due to ease of use and speed of analysis. In the first reaction step for the solid phase variant, the antibodies are adsorbed onto the solid phase. In this case, the reagents not bound to the solid phase are easily removed by washing. The test sample is incubated in the sensitized wells. In this case, immune complexes are formed on the surface of the solid phase. Unbound components are removed by washing. When the conjugate is added and bound to the immobilized immune complex, the active site of the enzyme remains available for subsequent interaction with the substrate. Incubation of the substrate in wells with immobilized conjugate leads to the development of a color reaction. This reaction can be stopped at the desired stage, the severity of staining can be assessed visually or by optical density.

The liquid-phase blocking indirect ELISA variant is closest to the neutralization test, which is the "gold standard" for determining the level of virus-neutralizing antibodies. In this variant of ELISA, the antigen is not attached to the substrate, its epitopes are the most accessible in space for the specific antibodies used, and this makes it possible to obtain the most approximate results to the neutralization reaction in the cell line; has high specificity and sensitivity, overall diagnostic accuracy [13].

In the last 6 years, the foot-and-mouth disease virus of the new O/ME-SA/Ind-2001 genotype has been widely spread [14, 15, 16]. The reference sequence of nucleotides and amino acids for strain O no. 2311/Zabaikalsky/2016 of genotype O/ME-SA/Ind-2001 is presented in GenBank [7] (Fig. 4).

Sharma G.K. et al. proposed a method for detecting antibodies against FMDV types A, O, Asia-1 using liquid-phase blocking indirect "sandwich" ELISA [17]. This technique makes it possible to identify a wide range of FMD antibodies, but the specificity for the FMDV genotype O/ME-SA/Ind-2001 is low (no more than 40%), and, therefore, accurate detection of immunoglobulins is not possible. It should be noted that this method involved the use of non-immune horse serum as a blocking component, which contains various protein compounds, including, possibly, homologous regions of amino acid sequences relative to antigenic determinants. To develop this method, monoclonal antibodies were used, the use of which increases the range of detection of various genetic lines of FMDV within one serotype, but at the same time, the specificity for the detection of antibodies against isolates/strains of a particular genotype, in particular O/ME-SA/Ind-2001, is significantly reduced. . Tetramethylbenzidine (TMB) was used as a dye, which is an aggressive carcinogen and an unstable component for carrying out a color reaction. Sulfuric acid, which is a dangerous compound, was used to stop the reaction.

In turn, the world market currently has European kits for the detection of antibodies against FMDV type O -IZSLER (Italy) and PrioCHECK (Netherlands) [18].

The method for determining the level of humoral immunity using the IZSLER kit is intended for the detection of FMD antibodies using a solid-phase direct "sandwich" ELISA variant. The manual for the proposed kits states that the percentage of inhibition in wells with a positive control of serum with a dilution of 1:10 should be ≥90%, for dilutions of 1:30 ->50%. Spectrophotometric readings must be ≥0.800 p.u. in the negative control wells.

The interpretation of the results is as follows:

- if the percentage of inhibition in the 1/10 and 1/30 dilution wells is ≥80%, then the sera are strongly positive;

- if the percentage of inhibition is in the range of 50-80% for serum dilutions of 1/10 and 1/30, then the sera are medium positive;

- if the percentage of inhibition is ≤50% for sera with dilutions of 1/10 and 1/30, then the sera are weakly positive.

Therefore, this technique allows for a semi-quantitative analysis of the level of humoral immunity of animals against FMDV.

The antibody titer is calculated as the extreme dilution of blood serum with a percentage of inhibition of at least 50%. The data is presented in the manufacturer's instructions.

The advantage of this kit is the use of monoclonal antibodies, however, they were obtained before the appearance of the new O/ME-SA/Ind-2001 genotype and do not allow high-precision detection of specific antibodies in animal blood sera. The specificity and sensitivity of this particular method for detecting antibodies against foot and mouth disease virus of the O/ME-SA/Ind-2001 genotype is no more than 40%, which is confirmed by the neutralization reaction data in the sensitive IB-RS-2 porcine kidney cell line. In other words, the test system used does not allow obtaining accurate results in relation to FMDV isolates and strains of the newly emerged genotype.

The closest prototype for the method we have developed is the method carried out using the PrioCHECK test system (Prionics Lelystad B.V., Netherlands) for the detection of antibodies to FMDV serotype O.

The components for the respective kit are shown below: Component 1 - Immunological plates.

Component 2 - Conjugate (30x). The diluted conjugate is not stable, prepare

immediately before use.

Component 3 - Dilution Buffer (5x).

Component 4 - Non-immune horse serum.

Component 5 - Demineralized water.

Component 6 - Wash Buffer (200x).

Component 7 - Reference serum 1 (positive) (liquid).

Component 8 - Reference serum 2 (less positive) (liquid).

Component 9 - Reference serum 3 (even less positive) (liquid).

Component 10 - Reference serum 4 (negative control) (liquid).

Component 11 - Chromogen Substrate (TMB).

Component 12 - Stop solution (1 N sulfuric acid solution). The data is reflected in the manufacturer's instructions.

The main steps of the ELISA reaction for the application of this method for determining the level of humoral immunity are presented below. Reconstituted non-immune horse serum, working buffer solution, ELISA buffer based on non-immune horse serum and buffer for dilution, dilution of the studied animal blood serum 1:5 are carried out. At the next stage of the analysis, the plate is incubated with the test and control blood sera for 1 hour at a temperature of 37±0.5°C, the wells of the plate are washed with buffer. Incubate the contents of the plate with the conjugate diluted to working dilution for 60 minutes. The wells of the plate are washed, incubated with a chromogen for 15 min at a temperature of 20 ± 0.5°C without access to light rays, the reaction is stopped with a stop solution, and optical densities are calculated using a tablet spectrophotometer-reader at a wavelength of 450 nm. Calculate the average value of OD _{450cp blank} contents of wells A1 and B1 with positive serum. OD _{450 cf blank} must be <0.350. Calculate the corrected OD ₄₅₀ for all samples by subtraction (OD _{450 corrected} =OD ₄₅₀ ×S - OD ₄₅₀ blank). Calculate the average value of the optical density for the contents of the wells G1 and H1 with negative serum (OD _{450 max} ). This value must be ≥1,000. If the OD _{450 max} is below 1,000, then the chromogen was most likely very cold. In this case, incubate for another 30 minutes.

The percent inhibition (PI) of reference and test sera is calculated according to the formula below:

PI=100-(OD _{450 corrected} /OD _{450 max} )×100

Calculate the average value of OD ₄₅₀ for wells with reference sera 2 and 3. The percentage of inhibition for S ₂ should be >60%, for S ₃ <40%.

This method allows you to get the results of the study in 4-5 hours. Diagnosticum includes a conjugate, which is a labeled monoclonal antibody against foot-and-mouth disease virus serotype O, therefore it is type-specific, but without a narrower differentiation.

A prototype method and a corresponding test system were developed at the end of the 90s of the XX century. At the same time, antibodies were obtained using FMDV antigens of various genotypes, except for the O/ME-SA/Ind-2001 genotype, which has been actively circulating since 2016. Isolates and strains of this line have significant differences from other lines of serotype O. Analyzing the nucleotide and amino acid sequence of the highly variable protein VP ₁ (gene 1D, approximately 3260 ... allowing to attribute the virus to a different genotype. Significant substitutions occurred in the epitopes of the surface proteins of the FMDV capsid, in particular, in the most variable regions (40–60, 130–160, and 190–213 a.a.) [14, 15, 16]. Based on the above, the prototype method with the test system used is not highly sensitive and specific to the FMDV strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001). Based on this, it is required to develop a highly sensitive, highly specific method for quickly determining the level of humoral immunity of animals to strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001) of the foot-and-mouth disease virus using a liquid-phase "sandwich" ELISA variant.

The prototype method involves the use of a test system based on a solid-phase competitive "sandwich" ELISA variant. However, this reaction format is further away from a neutralization reaction in terms of antigenic site trapping capabilities compared to the liquid phase blocking indirect sandwich ELISA.

The prototype version involves the use of native control animal blood sera, which reduces the safety of the components of the test system for a long period of time and there is a risk of contamination by microorganisms.

The prototype version does not provide for the control of the conjugate, which does not allow to obtain more reliable data on the optical density and titer of antibodies against FMDV.

In the prototype method, a direct conjugate is used, which is convenient to use, however, this increases the likelihood of noise background phenomena, which can lead to a decrease in the accuracy of determining the level of humoral immunity of animals against foot and mouth disease.

In the prototype method, tetramethylbenzidine TMB, which is a strong carcinogen, is used to display a color reaction, and sulfuric acid is used to stop the reaction, the working solution of which is prepared using a concentrate that negatively affects the mucous membranes of the respiratory and vision organs.

The specificity and sensitivity of this particular method for detecting antibodies against FMDV genotype O/ME-SA/Ind-2001 is no more than 45-50%, which is confirmed by the neutralization reaction data in the sensitive IB-RS-2 porcine kidney cell line. In other words, the test system used does not allow obtaining accurate results in relation to FMDV isolates and strains of the newly emerged genotype.

Considering the limitations found in the use of existing methods, including the prototype, to conduct a reliable study of the humoral immunity of animals after inoculation with FMD vaccines containing inactivated antigen of FMDV strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001, relevant is to develop a method for quickly determining the level of humoral immunity of animals to strain O No. 2311 / Zabaikalsky / 2016 of the foot-and-mouth disease virus using a liquid-phase "sandwich" ELISA variant, taking into account the above disadvantages.

At present, according to the literature data, no method has been found in the world for quantitative determination of the level of humoral immunity of animals against foot-and-mouth disease virus strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001 using a liquid-phase "sandwich" variant.

The aim of the invention is to develop a method for rapid quantitative determination of the level of humoral immunity of animals to strain O No. 2311/Zabaykalsky/2016 of foot-and-mouth disease virus (genotype O/ME-SA/Ind-2001) using a liquid-phase "sandwich" ELISA variant.

This goal is achieved by the fact that the proposed method involves the study of blood sera in dilutions with a minimum step to determine the titer of antibodies against strain O No. 2311/Zabaikalsky/2016 of the foot-and-mouth disease virus (genotype O/ME-SA/Ind-2001) using a liquid-phase "sandwich" - ELISA variant and the use of the following components: cultural inactivated FMDV strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001 (lyophilized); sensitizing antibodies - rabbit immunoglobulins G against FMDV strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001 (lyophilized); guinea pig G immunoglobulin detection antibodies against foot-and-mouth disease virus strain O No. 2311/Zabaikalsky/2016, genotype O/ME-SA/Ind-2001 (lyophilized); control 1 - lyophilized positive blood serum of cattle to foot-and-mouth disease virus strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001 (with a percentage of inhibition ≥75%); control 2 - lyophilized positive blood serum of cattle to foot-and-mouth disease virus strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001 (with the percentage of inhibition from 50% to 75%); control 3 - lyophilized blood serum of cattle, not containing antibodies to foot-and-mouth disease virus; lyophilized normal fetal bovine serum to block open binding sites at the bottom of the plate wells; anti-species conjugate - rabbit immunoglobulins against guinea pig IgG, conjugated with horseradish peroxidase. The proposed test system contains non-specific components: carbonate-bicarbonate buffer (pH 9.5-9.6) for antigen dilution (in the form of capsules), buffer solution concentrate (20x), chromogenic substrate ABTS (2,2-azino-di- (3-ethylbenzoaminosulfonic acid), "stop" solution - 1% solution of sodium dodecyl sulfate.

The goal is also achieved due to the fact that the liquid-phase blocking indirect "sandwich" ELISA method is closest to the neutralization reaction in vivo, since all antigenic sites of complete virions of FMDV strain O No. 2311/Zabaikalsky/2016 of the O/ME-SA/Ind genotype -2001 are in the free state in the liquid phase, open to binding with specific sensitizing and detection antibodies of rabbit and guinea pig, respectively. The reaction is based on the interaction of the studied animal blood serum and inactivated FMDV strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001 in the liquid phase in the wells of a round bottom plate with the formation of immune complexes. In parallel with this, the wells of a flat-bottomed plate are sensitized with rabbit immunoglobulins G against foot-and-mouth disease virus of the same genotype. After that, the sensitized wells are blocked with fetal calf serum, the resulting immune complexes are added from a round-bottom plate, incubated, and detector antibodies are added. The resulting complex immune complex is detected using antibodies against guinea pig IgG conjugated with horseradish peroxidase and a chromogenic substrate.

The technical result of the invention is to develop a fast (4 h), highly specific and sensitive method for quantitative determination of the level of humoral immunity of animals to strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001) of foot and mouth disease virus using sandwich" ELISA variant, due to the use of inactivated FMDV strain O No. 2311 / Zabaikalsky / 2016 genotype O / ME-SA / Ind-2001 and upon receipt of specific reaction components in the liquid phase.

Unlike the prototype, the developed method involves the use of cultured inactivated FMDV strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001, which is presented in a lyophilized state. This makes it possible to detect precisely antibodies against the FMDV of the specified genotype with high diagnostic sensitivity (more than 99%) and specificity (100%), which is more than 50-55% higher compared to the prototype. In addition, the freeze-dried component remains stable for at least 3 years than the antigen in the native state, which is an advantage in using this test system. The activity of the obtained antigen of foot-and-mouth disease virus strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001 is 1:4000.

Unlike the prototype, sensitizing and detection antibodies of a rabbit and a guinea pig are used, respectively, highly specific for the antigen of the foot-and-mouth disease virus strain O No. 2311/Zabaikalsky/2016 of the O/ME-SA/Ind-2001 genotype, since they were obtained using a highly purified antigen. The developed method involves the use of rabbit and guinea pig immunoglobulins G against foot-and-mouth disease virus strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001 in the form of a lyophilisate, which allows the antibodies to be in a stable state for at least 3 years with an activity of at least 1:12000 and 1:4000, respectively.

In contrast to the prototype blood serum of calves, controls 1 and 2 were obtained against a highly purified inactivated antigen of the foot and mouth disease virus strain O No. 2311/Zabaikalsky/2016 of the O/ME-SA/Ind-2001 genotype, which makes it possible to achieve high diagnostic specificity and sensitivity in the control. These components are presented in a lyophilized state, which allows them to be stored for at least 3 years while maintaining high activity in ELISA. For control 1 - not less than 1:4000, for control 2 - not less than 1:2500.

Unlike the prototype, fetal calf serum is used to block open binding sites, the protein components of which ensure complete closure of empty areas at the bottom of the immunological tablet. This component is also presented in the form of a lyophilisate, which allows it to be stored in this state for at least 3 years.

Unlike the prototype version, the developed method involves the use of antigen and conjugate control, which makes it possible to take into account background values and, thereby, obtain more reliable data on optical density and antibody titer against FMDV strain O No. 2311/Zabaikalsky/2016 genotype O/ME -SA/Ind-2001.

Unlike the prototype, the developed method involves the use of an indirect conjugate, which makes it possible to reduce the likelihood of noise background phenomena and obtain more reliable results for determining the level of humoral immunity against FMDV strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001 in sera animal blood. The activity of the conjugate is 1:2500.

The essence of the invention is reflected in the graphics:

Fig. 1 - Schematic diagram of a liquid-phase blocking indirect "sandwich" ELISA variant.

Fig. 2 - Spectral profile of FMDV antigen fractions O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001.

Fig. 3 - Electropherogram for inactivated FMDV strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001 in 12% polyacrylamide gel. Note: 1-3 - fractions No. 1-3 in the sucrose density gradient (50%); 4-8 - fractions No. 4-8 in the density gradient of sucrose (40%); 9-13 - fractions No. 9-13 in the density gradient of sucrose (30%); 14-18 - fractions No. 14-18 in the density gradient of sucrose (20%). A 100% antigen concentrate was tested.

Fig. 4 - Phylogenetic affiliation of strain O No. 2311 / Zabaikalsky / 2016 of the genotype O / ME-SA / Ind-2001 of the foot-and-mouth disease virus.

The essence of the invention is explained in the sequence listing, in which:

SEQ ID NO: 1 represents the nucleotide sequence of the VP ₁ protein gene of strain O No. 2311/Zabaikalsky/2016 of the genotype O/ME-SA/Ind-2001 of foot-and-mouth disease virus;

SEQ ID NO: 2 represents the amino acid sequence of the VP ₁ protein of the strain O No. 2311/Zabaikalsky/2016 of the genotype O/ME-SA/Ind-2001 of the foot-and-mouth disease virus.

The developed method for rapid quantitative determination of the level of humoral immunity of animals to strain O No. 2311/Zabaikalyekiy/2016 (genotype O/ME-SA/Ind-2001) of foot and mouth disease virus after vaccination using a liquid-phase "sandwich" ELISA variant is based on specific blocking of the foot and mouth disease virus antigen strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001) with antibodies contained in the test serum sample in the liquid phase. The test serum/antigen mixture is transferred to the wells of a plate previously immobilized with sensitizing antibodies. The presence of antibodies to foot-and-mouth disease virus strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001) in the test sample will be expressed in the formation of an immune complex and a decrease in the amount of free antigen that is bound by sensitizing antibodies immobilized on the plate. The antigen fixed in the wells interacts with detection antibodies, which are detected by reaction with an immunoperoxidase conjugate against guinea pig IgG and subsequent staining with the ABTS chromogenic substrate. The intensity of staining is inversely proportional to the concentration of specific antibodies in the test sample. The intensity of the color reaction is taken into account using a spectrophotometer-reader. Schematic diagram of the liquid-phase blocking indirect "sandwich"-ELISA version is shown in Fig.1.

Prior to the start of work, the preparation of working solutions is carried out.

Solution No. 1. Carbonate-bicarbonate buffer (CBC) solution, pH 9.5-9.6, for sensitization of tablets. The contents of the CBB capsule are dissolved in 100 cm ³ of distilled water and mixed thoroughly.

Solution number 2. The solution is used to prepare a working solution for inter-stage washing of plates and sample preparation for research, as well as the basis for preparing a working buffer solution for diluting detection antibodies and immunoperoxidase conjugate. To do this, the contents of the bottle with a concentrate (20x) of the buffer solution are poured into a measuring vessel, distilled water is added up to 2000 cm ³ and thoroughly mixed. The pH value of solution No. 2 should be in the range of 7.4-7.6.

Solution No. 3. The working buffer solution for dilution of detection antibodies and immunoperoxidase conjugate (pH 7.4-7.6) is prepared from solution No. 2 with the addition of 10% fetal cattle serum. To do this, add 4.0 cm ^{3 of} fetal bovine serum recovered from a lyophilized preparation by adding 4.0 cm ³ of distilled water to the contents of the vial with serum. Prepared immediately before use.

Before starting work, lyophilized preparations (antigen of foot and mouth disease virus strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001), conjugate, controls, fetal calf blood serum) are restored by adding 1 ml of distilled water. In the case of fetal serum - 4 ml of water.

The developed method includes the steps below. Tablet sensitization.

Suspension of sensitizing antibodies is prepared at a dilution of 1:12000 using solution No. 1 (see above). Into all wells of the immunological polystyrene 96-well plate, add 100 μl of a suspension of sensitizing antibodies, cover with a lid and incubate for 18±0.1 h at a temperature of (2-8)°C.

Washing the wells of the tablet.

Washing is carried out using automatic devices, or manually. At the same time, the contents of the wells of the tablet are removed, then the wells are filled with solution No. 2 (see above) and discarded by vigorous shaking. The procedure is repeated twice.

Immune binding in the liquid phase.

In parallel with the sensitization of the tablet, an immune antigen binding reaction is carried out with the test and control samples of blood serum. The following dilutions of animal blood sera are prepared: 1:12, 1:16, 1:24, 1:32, 1:45, 1:64, 1:90, 1:128, 1:181, 1:256, 1:362 , 1:512, 1:724, 1:1024 in solution No. 2. To do this, 0.047 cm ³ (47 μl) of buffer solution and 0.003 cm ³ (3 μl) of the sample . In wells H 1-6 add 0.05 cm ³ (50 μl) of buffer solution. The test and control samples are placed on the tablet according to table 1.

As control 1, positive blood serum of cattle against strain O No. 2311 / Zabaikalsky / 2016 (genotype O / ME-SA / Ind-2001) of foot-and-mouth disease virus (inhibition percentage ≥ 75%) is used, control 2 - positive blood serum of cattle against strain O No. 2311 / Zabaikalsky / 2016 (genotype O / ME-SA / Ind-2001) of foot and mouth disease virus (inhibition percentage from 50% to 75%), control 3 - normal calf serum.

Then, 0.05 cm ³ (50 μl) of the working dilution of the antigen in solution No. 2 is added to all wells of the tablet in accordance with a dilution of 1:4000. After mixing the contents of the wells of the tablet with a shaker or light shaking by hand, the plate is covered with a lid and incubated for 18±0.1 h at a temperature of (2-8)°C.

Antigen capture.

0.05 cm ³ (50 μl) of a mixture of control and test samples and antigen are added to the washed wells of the sensitized plate, the plate is closed with a lid and incubated for 1 hour at a temperature of (37±0.5)°C.

Washing the wells of the tablet.

Carried out as above. The procedure is repeated 4 times. Introduction of detection antibodies.

In all wells of the tablet, except for the wells with QC (conjugate control), where 0.05 cm ³ (50 μl) of solution No. 3 are added, 0.05 cm ³ (50 μl) of the working dilution of detection antibodies in solution No. 3 are added in accordance with the dilution of 1:4000, the tablet is covered with a lid and incubated for 1 hour at a temperature of (37±0.5)°C.

Washing the wells of the tablet.

Carried out as above. The procedure is repeated 4 times. Introduction of peroxidase conjugate.

0.05 cm ³ (50 µl) of the working dilution of the conjugate in solution No. 3 is added to all wells of the tablet in accordance with a dilution of 1:2500, the tablet is covered with a lid and incubated for 1 hour at a temperature of (37 ± 0.5) ° C .

Washing the wells of the tablet.

Carried out as above. The procedure is repeated 4 times. The manifestation of a color reaction.

0.05 cm ³ (50 µl) of the ABTS chromogenic substrate are added to all wells of the tablet, closed with a lid and incubated for 15-20 minutes at a temperature of (18-25)°C.

Reaction stop.

The reaction is stopped by adding to each well of the tablet 0.05 cm ³ (50 μl) "stop"-solution. Accounting for ELISA results.

The results of the analysis are taken into account in an instrumental way. Immediately after stopping the reaction, measure the optical density (OD) of the reaction products in each well at a wavelength of 405 nm using a spectrophotometer-reader for microplates with a vertical beam of light. The OD values (optical units (r.u.)) of the control and test samples are converted to the value of the percentage of inhibition (PI) according to the formula:

where, OD _{of the sample} is the average value of the optical density of the mixture of the test or control sample with the inactivated antigen of the FMDV strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001);

OP _KA - the average value of the optical density of the antigen control;

OD _QC - the average value of the optical density of the conjugate control.

A response is considered valid if it meets the following criteria:

- (OP _KA -OP _KK ) ≥0.4 p.u.;

- control 1 has a PI value>75%;

- control 2 has a PI value in the range of 50-75%;

- Control 3 has a PI<50%.

Samples demonstrating the value of PI≥50%> are considered positive, and the animals from which these blood serum samples are obtained are immune to foot and mouth disease caused by the virus strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001) . The value of PI<50% is negative, which indicates the absence of specific antibodies to foot-and-mouth disease caused by the virus strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001) in the blood serum of the examined animals. Based on the data obtained, take the last serum dilution for which the percentage of inhibition is ≥50%. This dilution is considered the titer of antibodies against foot-and-mouth disease caused by virus strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001). The level of humoral immunity in terms of quantity is expressed in log ₂ /ml.

As a result of the experiments, the conditions for setting up the reaction were optimized. The concentration of detection antibodies, determined in the indirect ELISA, was 1:4000, anti-species conjugate - 1:2500; the working dilution of sensitizing antibodies, determined by staging a liquid-phase blocking indirect "sandwich" ELISA variant, was 1:12000. Using the developed test system, we studied animal blood serum samples to determine the antibody titer and compared the results of the neutralization reaction in a sensitive monolayer porcine kidney cell line (IB-RS-2) [2].

The developed method allows testing simultaneously in the format of a 96-well plate up to 6 serum samples in dilutions from 1:12 to 1:1024.

As a working strain, strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001) of the foot-and-mouth disease virus was used.

Control of strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001) of foot-and-mouth disease virus for specificity.

The virus isolate, which served as a source for obtaining strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001) of the foot-and-mouth disease virus, was isolated from cattle on the territory of the Molodezhninskoye joint venture, Priargunsky district, Zabaikalsky Krai. Specificity was determined by RT-PCR followed by sequencing of the 1D gene corresponding to the VPi protein responsible for the variability of FMDV. According to the results of the study of the nucleotide sequence (presented in the appendix) of this gene, strain O No. 2311/Zabaikalsky/2016 was identified as belonging to the O/ME-SA/Ind-2001 genotype. This sequence is closest to the nucleotide sequence presented in GenBank under the number MG972584.1 [7]. The calculation was carried out in accordance with the Bayesian probability theory and bootstrap values. Specificity was also confirmed in the reaction of complement fixation with strain-specific serum. The concentration of the total viral protein in the studied antigen of the foot-and-mouth disease virus strain O No. 2311/Zabaikalsky/2016 was 2.85 µg/ml, the concentration of the 146S component was 1.83 µg/ml, the 75S component was 2.56 µg/ml, 12S was 0, 29 mcg/ml.

Compared with known strains, strain O No. 2311/Zabaikalsky/2016 has a higher infectious, antigenic and immunogenic activity.

The possibility of using FMD virus strain O No. 2311/Zabaikalsky/2016 for control of antigenic and immunogenic activity, as well as for the manufacture of biological products for the diagnosis and specific prevention of FMD serotype O has been experimentally confirmed.

Morphological features

The FMD virus strain O No. 2311/Zabaikalsky/2016 belongs to the family Picornaviridae, genus Aphthovirus, serotype A, genotype O/ME-SA/Ind-2001 and has morphological features characteristic of the FMD pathogen: icosahedral virion shape, size 23-25 nm . A virion consists of an RNA molecule enclosed in a protein shell. The protein shell consists of 32 capsomeres arranged in cubic symmetry.

Antigenic properties

According to its antigenic properties, the FMD virus strain O No. 2311/Zabaikalsky/2016 belongs to the O/ME-SA/Ind-2001 genotype. The virus is stably neutralized by homologous antiserum and does not exhibit hemagglutination activity. In ill animals, antibodies are formed in the blood serum, which are detected in ELISA and neutralization tests.

During hyperimmunization of guinea pigs, the concentrated antigen from the inactivated foot-and-mouth disease virus strain O No. 2311/Zabaikalsky/2016 induces the formation of virus-specific antibodies detected in RSK at a dilution of 1:2500.

The antigenic relationship of strain O No. 2311/Zabaikalsky/2016 with the available industrial strains of FMDV serotype O was studied in a cross-neutralization reaction. Strain O No. 2311/Zabaikalsky/2016 antigenically differs from industrial strains O No. 2222/Taiwan/2012, O No. 2147/Primorsky/2012, O No. 2212/Primorsky/2014, O No. 2047/Saudi Arabia/2008.

The results of studies in the neutralization reaction are presented in table 2. Antigenic correspondence (r ₁ ) was 0.12 for O No. 2222/Taiwan/2012, O No. 2147/Primorsky/2012 - 0.27, O 1734/Primorsky/2000 - 0 .29, O No. 2356/Pakistan/2018 - 0.19. If r ₁ ≥0.3, the field isolate and production strain are closely related, and the vaccine from the production strain will protect against the epizootic field virus, if r ₁ <0.3, the field isolate differs from the production strain, and the vaccine from this strain does not protect from an epizootic field virus.

Biotechnological characteristics

Strain O No. 2311/Zabaikalsky/2016 is reproduced in transplanted monolayer cell lines of the kidney of the Siberian mountain ibex (PSGK-30), the kidney of the pig IB-RS-2, the kidney of the newborn Syrian hamster VNK-21, and within 12-15 hours of incubation of the virus in these cell cultures accumulate from 6.50 to 7.75 lg TCD ₅₀ /cm ³ .

Genotaxonomic characteristics.

Strain O No. 2311/Zabaikalsky/2016 of FMDV type O is an RNA-containing virus with a molecular weight of 7×10 ⁶ Da.

Nucleic acid is represented by a single-stranded linear molecule with a molecular weight of 2.8×10 ⁶ Da. The virion has a protein coat consisting of four structural proteins VP ₁ , VP ₂ , VP ₃ and VP ₄ .

The main antigenic protein is VP ₁ . The virion contains approximately 31.5% RNA and 68.5% protein. Virion RNA is infectious and is involved in the formation of precursor proteins in infected cells. The precursors, in turn, are cleaved to form more stable structural and non-structural virus polypeptides. Of the 8 non-structural polypeptides that accumulate in infected cells, one (VP _66a ) is an RNA-dependent RNA polymerase involved in RNA replication of new virions.

physical properties.

The weight of the virion is 8.4×10 ^-18 g. The floating density of the virions in the sucrose solution is 1.45 g/cm ³ .

Resilience to external factors.

Strain O No. 2311/Zabaikalsky/2016 is resistant to ether, chloroform, freon, acetone and other organic solvents and detergents. Most stable at pH 7.4-7.8. Shifts in pH, both in the acidic and alkaline directions, lead to virus inactivation. Sensitive to formaldehyde, UV radiation, γ-irradiation, high temperatures.

Additional signs and properties. Immunogenic activity - immunogenic as part of an inactivated vaccine.

Antigenic activity - the introduction of the antigen of the foot-and-mouth disease virus strain O No. 2311 / Zabaikalsky / 2016 of the O / ME-SA / Ind-2001 genotype to guinea pigs induces the formation of virus-specific antibodies.

Reactogenicity - does not possess reactogenic properties.

Pathogenicity - pathogenic for artiodactyl animals, newborn mice, guinea pigs.

Virulence - virulent for naturally susceptible animals with contact, aerosol and parenteral infection.

Stability - retains the original biological properties when passaged in sensitive biological systems for 5 passages (observation period).

The essence of the invention is illustrated by examples of its use, which do not limit the scope of the invention.

Example 1. Obtaining an inactivated foot-and-mouth disease virus strain O No. 2311 / Zabaikalsky / 2016

To obtain inactivated FMDV strain O No. 2311/Zabaikalsky/2016 for diagnostic purposes, suspension cultivation of the virus was carried out in the VNK-21 cell line for 12-14 hours to obtain a viral suspension with an infectious activity titer of at least 6.5 lg TCD ₅₀ /cm ³ . The resulting viral suspension was inactivated with aminoethylethyleneimine and clarified with polyhexamethyleneguanidine.

The inactivated culture liquid containing the FMD virus antigen was clarified for 30 min. at 6000 rpm and 4°C in a Beckman J2-21 or Avanti J-26 XP centrifuge (Beckman Coulter, USA). The supernatant was collected.

PEG-6000 was added to the supernatant to a final concentration of 8% and NaCl to a final concentration of 0.85%, vigorously mixed and kept at a temperature of 4±2°C for 18-24 hours. The virus suspension was precipitated at 6000 rpm for 60 minutes, the precipitate was dissolved in 1/15 M PBS solution, concentrating approximately 100 times (1/100 of the original volume).

Chloroform (50%) was added to the resulting precipitate, vigorously stirred, and fractionated for 30 min. at 3000 rpm. The upper water fraction containing the antigen of the foot and mouth disease virus strain O No. 2311/Zabaikalsky/2016 was taken. A small amount (50-100 μl) was transferred into a cryovial and frozen at a temperature of minus 20±2°C for subsequent electrophoretic analysis in a polyacrylamide gel.

At the next stage, the 146S component of foot and mouth disease virus strain O No. 2311/Zabaikalsky/2016 was obtained (this component is immunogenic and the most important in animal immunization, since it is a complete virion).

Before being placed in the sucrose gradient, the FMDV antigen was treated with NP-40 detergent at a concentration of 1.5%.

To isolate the 146S component, a linear gradient of sucrose was prepared with concentrations of 10, 20, 30, 40, and 50%. The FMDV antigen, strain O No. 2311/Zabaikalsky/2016, treated with NP-40, was poured into centrifuge tubes in 13-15 ml increments, then sucrose solutions were sequentially layered, starting with a 10% and ending with a 50% solution. The tubes were placed in metal centrifuge cups and, after careful balancing, were unscrewed at a temperature of 4°C for 4 hours at a speed of 25,000 rpm.

Fractionation of the sucrose gradient was performed using a peristaltic pump, taking fractions of 1 ml into separate tubes.

When using a peristaltic pump, the sucrose gradient tube was first visually evaluated. The protein band containing the 146S component is located in approximately 30% sucrose layer and stands out as opalescence. Fractions with 50, 40, 30 and 20% sucrose were taken. The results of electrophoresis in 12% polyacrylamide gel under denaturing conditions are shown in FIG. 3.

The 146S component was then reprecipitated by ultracentrifugation for 4 hours at 25,000 rpm. and a temperature of 4±0.5°C and the pellet was resuspended in 1/15 M phosphate buffer solution.

The results of a spectrometric study of the obtained fractions of the FMDV antigen of strain O No. 2311/Zabaikalsky/2016 in the form of an antigenic profile are shown in Fig. 2.

Example 2. Obtaining hyperimmune rabbit blood serum (sensitizing antibodies)

To obtain specific blood sera against foot-and-mouth disease virus strain O No. 2311/Zabaikalsky/2016, clinically healthy rabbits of average fatness weighing 2.5-3.0 kg were used.

For hyperimmunization of animals, a concentrated purified antigen of foot and mouth disease virus strain O No. 2311/Zabaikalsky/2016, obtained as described in example 1, was used, from which an emulsion was prepared using Montanide ISA-61 VG oil adjuvant (in the ratio of adjuvant/antigen = 60/40 by weight ). The resulting vaccine was injected into the muscle of the hind limbs of the rabbit on days 0, 21 and 42 in a volume of 0.5 cm ³ . 7 days after the last immunization, the rabbits were totally bled and received blood serum - containing sensitizing antibodies, which was freeze-dried and stored at a temperature of 4-8°C.

Example 3. Obtaining hyperimmune guinea pig blood serum (detector antibodies)

For hyperimmunization of guinea pigs, an emulsion of a purified preparation of a concentrated antigen of the strain O No. 2311 / Zabaikalsky / 2016 of foot-and-mouth disease virus, obtained as described in example 1, was used. The preparation of the vaccine and the immunization scheme are the same as in example 2.

Example 4. Determination of the activity of the antigen of the strain O No. 2311 / Zabaikalsky / 2016 of the foot-and-mouth disease virus, sensitizing and detection antibodies

Individual sera samples were tested for activity and specificity in a liquid-phase blocking indirect "sandwich" variant of the ELISA. The optimal working dilutions of these components were selected to obtain reliable results in ELISA. The obtained analysis data are presented in tables 3, 4, 5.

The following dilutions of sensitizing antibodies were tested for analysis: 1:8000, 1:10000, 1:12000, 1:14000. The dilutions of the remaining components were constant: for antigen - 1:4000, for detection antibodies - 1:4000, conjugate - 1:2500. Serums specific to certain strains of FMDV with an activity of 1:256 were used as control sera for analysis. Their table 3 shows that when the dilution of sensitizing antibodies 1:12000 achieved a reliable result (1:256 or 8 log ₂ /ml). At lower antibody dilutions, high background values were observed, which did not allow a correct result to be obtained. At a higher dilution, a decrease in the detectable value of antibody titer in the blood sera of animals was observed. In the study of non-specific and negative sera, negative results were noted, which indicated the high specificity of the developed method.

For analysis, the following dilutions of detection antibodies were studied: 1:2000, 1:3000, 1:4000, 1:5000. The dilutions of the remaining components were constant: for antigen - 1:4000, for sensitizing antibodies -1:12000, conjugate - 1:2500. Serums specific to certain strains of FMDV with an activity of 1:256 were used as control sera for analysis. From Table 4 it follows that a 1:4000 dilution of the detection antibodies achieved a reliable result (1:256 or 8 log ₂ /ml). At lower antibody dilutions, high background values were observed, which did not allow a correct result to be obtained. At a higher dilution, a decrease in the detectable value of antibody titer in the blood sera of animals was observed. In the study of non-specific and negative sera, negative results were noted, which indicated the high specificity of the developed method.

The following antigen dilutions were examined for analysis: 1:2000, 1:3000, 1:4000, 1:5000. The dilutions of the other components were constant: for detection antibodies - 1:4000, for sensitizing antibodies -1:12000, conjugate - 1:2500. Serums specific to certain strains of FMDV with an activity of 1:256 were used as control sera for analysis. Their table 5 shows that when the antigen was diluted 1:4000, a reliable result was achieved (1:256 or 8 log ₂ /ml). At lower antibody dilutions, high background values were observed, which did not allow a correct result to be obtained. At a higher dilution, a decrease in the detectable value of antibody titer in the blood sera of animals was observed. In the study of non-specific and negative sera, negative results were noted, which indicated the high specificity of the developed method.

Thus, in the course of laboratory studies it was proved that the working dilutions of the antigen of the strain O No. 2311 / Zabaikalsky / 2016 of the foot-and-mouth disease virus, sensitizing and detection antibodies were 1:4000, 1:12000, 1:4000, respectively.

Example 5. Application of the method for rapid quantitative determination of the level of humoral immunity of animals to strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001) of foot-and-mouth disease virus after vaccination using a liquid-phase "sandwich" ELISA variant

We studied 6 blood sera of cattle and 6 blood sera of pigs obtained after immunization of animals with FMD cultural inactivated emulsion vaccines containing the 146S component of FMDV strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001 at least 3.0 μg /dose.

The values of the antibody titer were determined using the developed method, and the serum data were studied in the neutralization reaction in the sensitive monolayer cell line IB-RS-2, in accordance with the recommendations of the OIE Guidelines (OIE) [2]. The results obtained are shown in table 6, from which it follows that the degree of correlation between the results of the developed method (the proposed invention) and the classical method (neutralization reaction) is 100%.

Example 6. Determination of the degree of correlation of the results of the developed method with the data of the neutralization reaction and the ELISA test system (PrioCHECK) in the study of a large number of animal sera

For analysis, we used 595 blood sera of animals containing antibodies against the 146S component of FMDV strain O No. 2311/Zabaikalsky/2016 with specific antibody titers 1:16-1:1024. These sera were studied in the neutralization reaction in accordance with the recommendations of the OIE Guidelines (OIE), using the ELISA test system (PrioCHECK) and using the developed method in the liquid-phase blocking indirect "sandwich" ELISA version (the proposed invention) to determine the level of humoral immunity against foot-and-mouth disease virus strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001).

It was found that the data (n=595) obtained using the developed test system correlated with the data of the neutralization reaction by 99.01%) for the blood sera of animals with an antibody titer of 1:16 (n=85), by 99.14% for animal blood sera with antibody titer 1:32 (n=85), by 99.24% for animal blood sera with antibody titer 1:64 (n=85), by 99.09% for animal blood sera with antibody titer 1 :128 (n=85), 99.52% for animal blood sera with antibody titer 1:256 (n=85), 99.58% for animal blood sera with antibody titer 1:512 (n=85) , by 99.80% for the blood sera of animals with an antibody titer of 1:1024 (n=85). The results obtained testified to the high degree of accuracy of the developed rapid quantitative determination of the level of humoral immunity of animals to the strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001) of foot-and-mouth disease virus using the liquid-phase "sandwich" ELISA after vaccination according to compared to the neutralization reaction.

When examining these sera using the PrioCHECK test system in ELISA (prototype), it was found that the detected values of animal antibody titers against the antigen of strain O No. -55% lower compared to the data of the neutralization reaction and the developed method (the proposed invention).

Thus, the developed method makes it possible to reliably quantify the level of humoral immunity of animals to strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001) of foot-and-mouth disease virus using a liquid-phase "sandwich" ELISA variant.

Example 7. The study of animal blood sera against other strains of the O/ME-SA/Ind-2001 genotype using the developed method

To prove that the developed method is not only highly sensitive and specific, but also universal for quantitative determination of the level of humoral immunity of animals against various strains of the FMDV genotype O/ME-SA/Ind-2001, a study of 142 blood sera against the antigen of industrial strains of the same genotype. It was found that the degree of correlation with the results of the neutralization reaction was 99.01-99.79%, which allows us to conclude that the developed method can be used to determine the antibody titer against the FMDV antigen of various strains belonging to the O/ME-SA/Ind- genotype. 2001.

Example 8. Determination of diagnostic parameters of the developed method for rapid quantitative determination of the level of humoral immunity of animals to strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001) of foot-and-mouth disease virus using liquid-phase "sandwich" ELISA

To study the developed method (the proposed invention) for the rapid quantitative determination of the level of humoral immunity of animals to strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001) of the foot-and-mouth disease virus using a liquid-phase "sandwich" ELISA variant, standard diagnostic tests were studied. indicators. To determine the sensitivity of the proposed test system, 520 animal sera were analyzed, which were known to be positive according to the neutralization test in the sensitive IB-RS-2 porcine kidney cell line. The antibody titer for these blood sera was in the range of 1:32-1:1024. The ELISA was performed as described above. Using the developed method (the proposed invention), it was determined that out of 520 blood serum samples, 519 were identified as positive, and 1 as negative (titer 1:16). To study the specificity of the method, 305 negative blood sera were tested. As a result of the study using the developed method (the proposed invention), it was determined that all 305 sera samples were negative. Using the statistical methods of analysis presented above, it was determined that the diagnostic sensitivity (DSe) was 99.81% (in the 95% confidence interval: 98.94-100.00%), the diagnostic specificity (DSp) was 100%) (in 95% confidence interval: 98.80-100.0%), k-test - 0.997; positive predictive value (PPV) - 100%, negative predictive value (NPV) - 99.67% (95% confidence interval: 97.73-99.95%), overall accuracy (DAc) - 99.88% ) (in 95% confidence interval: 99.33-100.00%) (Table 7).

Thus, the proposed "Method for rapid quantitative determination of the level of humoral immunity of animals to strain O No. 2311 / Zabaikalsky / 2016 (genotype O / ME-SA / Ind-2001) of foot-and-mouth disease virus using a liquid-phase" sandwich "ELISA variant" is highly specific and sensitive, allowing for a quick analysis of animal blood sera within 4 hours with a quantitative determination of the level of antibodies after vaccination against foot-and-mouth disease caused by foot-and-mouth disease virus strain O No. 2311 / Zabaikalsky / 2016 (genotype O / ME-SA / Ind-2001).

Sources of information taken into account when compiling the description of the invention to the application for a patent of the Russian Federation for the invention "Method for rapid quantitative determination of the level of humoral immunity of animals to strain O No. 2311/Zabaikalsky/2016 (genotype O/ME-SA/Ind-2001) of foot and mouth disease virus using a liquid-phase "sandwich" ELISA variant":

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6.O.I.E. Manual of diagnostic tests and vaccines for terrestrial animals. - Paris, 2018. - Chapter 2.1.8.

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8. Gulenkin V.M. Economic efficiency of preventive vaccination of animals against foot-and-mouth disease on the territory of the Russian Federation / V.M. Gulenkin // Proceedings of the Federal Center for Animal Health. - Vladimir, 2012. - T. 10. - S.31-41.

9. Russian patent RU 2 658 608, 02.11.2017 according to IPC C12N 7/00 A61K 39/135. Strain O No. 2311/Zabaikalsky/2016 of the Aphtae epizooticae type O foot-and-mouth disease virus for the manufacture of biological products and specific prevention of foot-and-mouth disease type O.

10. Russian patent RU 2665849, 12/15/2017 IPC C12N 7/00 Inactivated emulsion vaccine against FMD type O.

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Sequence listing

<110> Federal State Budgetary Institution "Federal Center

Animal Health Protection (FSBI ARRIAH) Federal Governmental

Budgetary Institution "Federal Center for Animal Health"

(FGBI "ARRIAH")

<120> A method for rapidly quantifying the level of humoral

immunity of animals to strain O No. 2311/Zabaikalsky/2016 genotype

O/ME-SA/Ind-2001 FMDV after vaccination with

liquid phase "sandwich" ELISA

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<400> 1

ACCACCTCCA CAGGTGAGTC CGCTGATCCC GTGACCACCA CCGTTGAGAA CTACGGTGGA 60

GAGACACAGG TCCAGAGACG TCAACACACC GACGTTTCTT TCATTTTGGA CAGATTTGTG 120

AAAGTAACAC CGAAAGACCA AATCAATGTG TTGGACCTGA TGCAAACCCC TGCTCACACT 180

TTGGTAGGCG CACTCCTCCG CACCGCCACT TACTACTTCG CAGACCTAGA AGTGGCAGTG 240

AAGCACGAGG GCAACCTCAC CTGGGTCCCG AACGGGGCGC CCGAGGCGGC GCTGGACAAC 300

ACCACCAACC CGACGGCCTA CCACAAGGCA CCGCTCACCC GTCTTGCTCT GCCTTACACA 360

GCACCACACC GTGTTCTGGC TACCGTTTAC AACGGGAACT GCAAGTATGG CGAGGGCGCT 420

GTGACCAACG TGAGGGGTGA CTTGCAAGTG TTGGCTCAGA AGGCAGCAAG AACGCTGCCC 480

ACCTCCTTTA ACTACGGTGC CATCAAGGCT ACCCGGGTGA CTGAACTGCT TTACCGCATG 540

AAGAGGGCCG AAACATACTG CCCTCGGCCT CTGCTGGCCA TTCACCCGGA ACAAGCCAGA 600

CACAAGCAGA AGATTGTGGC ACTGTGAAA CAGTTGTTG 639

<210> 2

<211> 213

<212> PRT

<213> foot-and-mouth disease virus

<400> 2

1 Thr Thr Ser Thr Gly Glu Ser Ala Asp Pro Val Thr Thr Thr Val 15

16 Glu Asn Tyr Gly Gly Glu Thr Gln Val Gln Arg Arg Gln His Thr 30

31 Asp Val Ser Phe Ile Leu Asp Arg Phe Val Lys Val Thr Pro Lys 45

46 Asp Gln Ile Asn Val Leu Asp Leu Met Gln Thr Pro Ala His Thr 60

61 Leu Val Gly Ala Leu Leu Arg Thr Ala Thr Tyr Tyr Phe Ala Asp 75

76 Leu Glu Val Ala Val Lys His Glu Gly Asn Leu Thr Trp Val Pro 90

91 Asn Gly Ala Pro Glu Ala Ala Leu Asp Asn Thr Thr Asn Pro Thr 105

106 Ala Tyr His Lys Ala Pro Leu Thr Arg Leu Ala Leu Pro Tyr Thr 120

121 Ala Pro His Arg Val Leu Ala Thr Val Tyr Asn Gly Asn Cys Lys 135

136 Tyr Gly Glu Gly Ala Val Thr Asn Val Arg Gly Asp Leu Gln Val 150

151 Leu Ala Gln Lys Ala Ala Arg Thr Leu Pro Thr Ser Phe Asn Tyr 165

166 Gly Ala Ile Lys Ala Thr Arg Val Thr Glu Leu Leu Tyr Arg Met 180

181 Lys Arg Ala Glu Thr Tyr Cys Pro Arg Pro Leu Leu Ala Ile His 195

196 Pro Glu Gln Ala Arg His Lys Gln Lys Ile Val Ala Pro Val Lys 210

211 Gln Leu Leu 213

<---

Claims (3)

1. A method for rapid quantitative determination of the level of humoral immunity of animals to strain O No. 2311 / Zabaikalsky / 2016, genotype O / ME-SA / Ind-2001, foot-and-mouth disease virus using a liquid-phase “sandwich” ELISA variant, involving the use of a cultured inactivated foot-and-mouth disease virus strain O No. 2311 / Zabaikalsky / 2016 genotype O / ME-SA / Ind-2001 - lyophilized; sensitizing antibodies - rabbit immunoglobulins G against FMDV strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001 - lyophilized; detection antibodies - guinea pig immunoglobulins G against FMDV strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001 - lyophilized; control 1 - lyophilized positive blood serum of cattle to foot-and-mouth disease virus strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001, with a percentage of inhibition ≥75%; control 2 - lyophilized positive blood serum of cattle to foot-and-mouth disease virus strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001, with a percentage of inhibition from 50% to 75%; control 3 - lyophilized blood serum of cattle, not containing antibodies to foot-and-mouth disease virus; lyophilized normal fetal bovine serum to block open binding sites at the bottom of the wells of the tablet; anti-species conjugate - rabbit immunoglobulins against guinea pig IgG, conjugated with horseradish peroxidase, carbonate-bicarbonate buffer pH 9.5-9.6, for antigen dilution, in the form of capsules, concentrate - 20x buffer solution, chromogenic substrate ABTS - 2.2- azino-di-3-ethylbenzoaminosulfonic acid, "stop" solution - 1% solution of sodium dodecyl sulfate; and a modified formula is used to determine the percentage of inhibition:

in this case, the highest dilution of the studied blood serum, for which the value of the percentage of inhibition is ≥50%, is taken as the antibody titer; the level of humoral immunity is expressed in log ₂ /ml. 2. The method according to claim 1 allows you to quickly analyze animal blood sera within 4 hours with a quantitative determination of the level of humoral immunity after vaccination against foot-and-mouth disease caused by the virus strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001 and determine their titer in the range of 1:12-1:1024 with a reliability of at least 99%, a diagnostic sensitivity of 99.81%, and a diagnostic specificity of 100%.

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