RU2791614C1 - Test system based on liquid phase blocking indirect "sandwich" elisa for determination of antibody titer against fmdv strain o n2047/saudi arabia/2008 genotype o/me-sa/panasia2 in animal blood sera - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to the creation of a test system for a liquid-phase blocking indirect "sandwich" ELISA variant for determining the titer of antibodies against FMDV strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2 in animal blood sera. The advantage of the developed test system is a long shelf life (up to 3 years) due to the production of freeze-dried immunospecific and non-immunospecific components.

EFFECT: proposed test system provides the creation of a modern, highly specific and sensitive test system designed to quantify the antibody titer against FMDV strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2 in a wide range of 4.0-10, 5 log2 (1:16-1:1448) for subsequent analysis of the immune status of animals in relation to FMD.

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Description

The invention relates to the field of veterinary virology and biotechnology, in particular the creation of a test system for a liquid-phase blocking indirect "sandwich" ELISA variant for determining the titer of antibodies against foot-and-mouth disease virus strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2 in sera animal blood.

Foot and mouth disease is a highly contagious viral disease of artiodactyl animals, which is characterized by a high incidence, which entails restrictions on the international trade in livestock and its products. The main clinical signs of the disease are fever, lameness, aphthae in the mouth, feet and nipples. Young animals have a higher mortality due to myocarditis compared to adults. Outbreaks of foot and mouth disease often occur in the countries of the Middle East and Asia, Africa [1, 2].

FMDV is a representative of the genus Aphthovirus of the family Picornaviridae and has 7 different serotypes: O, A, Asia-1, C and SAT-1, SAT-2, SAT-3 [2]. The genome encodes 4 structural proteins VP ₁ , VP ₂ , VP ₃ , VP ₄ . The GH loop of the VP ₁ protein has been identified as the main antigenic site. The foot-and-mouth disease virus RNA also carries information about 8 non-structural proteins (L-protease, 2A, 2B, 2C, 3A, 3B, 3C-protease and 3D-RNA-dependent RNA polymerase). FMDV has a very high mutation rate due to the lack of a repair mechanism for the viral RNA-dependent RNA polymerase. Antigenic variants are caused by various amino acid mutations that lead to difficulties in the fight against FMD [1, 2].

In RNA viruses, genome variation is favored by high mutation rates during virus replication, and emerging lineages and sublines are driven by mutation and recombination. Recombination is another important process that determines the biology and evolution of viruses. It is the exchange of genetic material between two non-segmented RNA genomes. It has been shown that genetic recombination occurs between viruses of the same serotype [3, 4]. Intratypic recombination occurs frequently and recombination events in FMD appear to occur more readily in the 3' portion of the genome than in regions encoding structural viral proteins. Mutations resulting from recombination lead to the exchange of genetic material and the formation of new antigenic variants, which can avoid immune pressure and lead to changes in cell tropism and host range [2, 5, 6]. 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 will acquire the ability to penetrate cells using alternative receptors [6].

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 host cells [6, 7]. Mutations in the genes encoding capsid proteins can lead to antigenic changes and the emergence of new genetic lines [6].

The most variable protein is FMDV VP ₁ , since it accounts for at least 90% of mutations in all structural genes. The most variable regions are regions 40–60, 130–160, and 190–213 a.a. [7]. 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 [8, 9, 10]. The variability of this region enables FMDV to interact with receptors of different cell types and facilitates the transition from one host species to another [9].

In 2008, the foot-and-mouth disease virus, which belongs to the O/ME-SA/PanAsia2 genotype, became widespread in Saudi Arabia. In March 2008, the isolate O/SAU/5/2008 was isolated from a calf in this country. The nucleic acid ID gene of this virus was analyzed using two primer systems O-1C244F/EUR-2B52R and O-1C272BF/EUR-2B52R. A 639 bp cDNA fragment of the obtained isolate was isolated. Based on the results of sequencing, it was determined that the obtained isolate is 99.53% similar to representatives of the O/ME-SA/PanAsia2 genotype. The nucleotide sequences of the ID gene and amino acids of the VP1 protein for the indicated isolate were published in the GenBank database on June 14, 2021 under the number MT 443784.1. It should be noted that no genetic subline has been assigned to this isolate. A narrower division by genetics began to be carried out for FMDV isolates isolated starting in 2009.

At the Federal State Budgetary Institution "ARRIAH", this isolate was adapted to various sensitive cell lines, including the monolayer and suspension transplantable cell line of the kidney of a newborn Syrian hamster VNK-21, and strain O No. 2047 / Saudi Arabia / 2008 of the O / ME-SA genotype was obtained /PanAsia2. This strain was deposited in the All-Russian State Collection of Exotic Types of Foot and Mouth Virus and Other Animal Pathogens (GKSM) of the Federal State Budgetary Institution “ARRIAH” (certificate No. 33 on the deposit of the strain “O No. 2047/Saudi Arabia/2008” of the O foot and mouth disease virus serotype for the purposes of the patent procedure dated December 24 .2021) under the registration number: strain VYa O No. 2047/Saudi Arabia/2008. Fig. 4.

Immunization is an effective tool in the fight against disease. 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" has developed a vaccine, which includes the antigen of the FMD virus of this strain, this genotype.

There was a need to create highly specific and highly sensitive diagnostic test systems that would allow the detection of antibodies against FMDV strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2.

Protection against FMD after vaccination is determined by the level of specific antibodies in the blood serum of animals [11, 12, 13, 14]. The level of humoral immunity of the vaccinated livestock is determined by detecting antibodies specific to the capsid or structural proteins of the foot-and-mouth disease virus. In accordance with the recommendations of the OIE (OIE) [2], neutralization reaction and enzyme immunoassay (ELISA) are used to determine the immune status of animals. The neutralization reaction is considered a sensitive and specific method, however, the analysis has a number of the following limitations: 1) a long reaction time (at least 3 days), 2) the high cost of the procedure associated with the use of a sensitive cell line, the presence of a CO ₂ incubator, 3 ) involves the use of live foot-and-mouth disease virus, which is a biological agent of hazard group II.

In turn, ELISAs have 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 [15, 16]. Based on this, it is advisable to use ELISA to determine the antibody titer in vaccinated animals.

Allocate 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.

There is an indirect variant of ELISA, which has significant advantages over the direct one. The advantages of this variant of the method include the following: 1) the presence of a wide variety of labeled secondary antibodies, 2) universality (many primary antibodies can be obtained from the same species, and the same labeled secondary antibody can be used for detection), 3) maximum immune the activity of the primary antibody is preserved because it is not labeled, 4) increased sensitivity, since each primary antibody contains several epitopes that can be associated with a labeled secondary antibody, which allows to enhance the detected signal. The limitations of the method include the possibility of cross activity with a secondary antibody, which can lead to the appearance of a nonspecific signal, as well as the presence of an additional reaction step [4, 15, 17].

Of great importance in laboratory work is the "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 they are designed to capture the antigen. The second antibodies - detector, are necessary for the detection of the antigen. This ELISA variant has the following advantages:

- high specificity of the analysis;

- suitable for the study of field samples of varying degrees of purification;

- high reaction sensitivity.

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 has a number of the following advantages: 1) the analysis of all modifications of this analysis is closest to the classical neutralization reaction, since the antigen particles are not attached to the substrate; 2) antigen epitopes are most accessible in space for specific antibodies, and this allows obtaining the most approximate results to the neutralization reaction in the cell line; 3) has high rates of analytical and diagnostic sensitivity, specificity and overall accuracy.

Sharma G.K. et al. (2015) proposed a method for detecting antibodies against FMDV types A, O, Asia-1 using liquid-phase blocking indirect "sandwich" ELISA [18]. This technique made it possible to identify a wide range of anti-FMD antibodies, but at the same time, the specificity for FMDV of the O/ME-SA/PanAsia2 genotype is not more than 50%, and, therefore, the accurate detection of immunoglobulins G 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. The authors of the test system used monoclonal antibodies, the use of which increases the spectrum 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/PanAsia2, 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.

The above test systems are not presented on the market as a kit for the detection of FMD antibodies in animal blood sera and remain as diagnosticums inaccessible to the general consumer. In turn, the world market currently has European kits for the detection of antibodies against FMDV type O - IZSLER (Italy) and PrioCHECK (Netherlands).

The test system in the IZSLER kit is intended for the detection of FMD antibodies using a solid-phase direct "sandwich" ELISA. The components of the IZSLER kit (Instituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna Brescia, Italy The Pirbright Institute) are as follows:

1. Sealed microplates sensitized with inactivated FMDV type O.

2. Conjugate (monoclonal antibodies against FMDV type O, conjugated with peroxidase).

3. Serum positive control against FMDV type O.

4. Negative control.

5. Buffer solution for ELISA - phosphate-saline buffer with tween-20 (FSB-Tween).

6. Buffer for dilution of blood serum and conjugate.

7. Substrate - TMB chromogen solution.

8. Solution to stop the reaction - 0.6N solution of sulfuric acid. The advantage of this kit is the use of monoclonal antibodies, however, they were obtained earlier than 2008 (the period when strain O No. 2047 / Saudi Arabia / 2008 appeared, since 2014 it began to be used as a production strain) and cannot detect with high reliability and accuracy specific antibodies in animal blood sera. The specificity and sensitivity of the specified test system for the detection of antibodies against FMDV genotype O/ME-SA/PanAsia2 is below 50%, which is confirmed by the neutralization reaction data in the sensitive continuous monolayer porcine kidney cell line IB-RS-2. In other words, the test system used does not allow obtaining accurate results specifically in relation to FMDV isolates and strains of the newly appeared genotype.

The closest prototype for the test system developed by us is the PrioCHECK test system (Prionics Lelystad B.V., Netherlands) for detecting antibodies to FMDV type O.

The accessories 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 - TMB chromogen substrate.

Component 12 - Stop solution - 1 N sulfuric acid solution. Data on the configuration of the test system are reflected in the manufacturer's instructions.

Diagnosticum is easy to use and allows you to get the results of the study in 5-6 hours. This test system 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 test system was developed at the end of the 90s of the XX century. and obtained corresponding antibodies against FMDV antigens of various genotypes, created using FMDV antigens of various genotypes, except for the O/ME-SA/PanAsia2 genotype, which has been actively circulating since 2008. 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 peptide of foot-and-mouth disease virus VP], the scientists concluded that there are a sufficient number of significant substitutions to attribute the virus to a different genotype. Based on the above, the prototype test system is not highly sensitive and specific to the FMDV genotype O/ME-SA/PanAsia2. This test system detects antibodies to this strain by no more than 60%. Based on this, it is required to develop a highly sensitive, specific test system for the detection and quantitative analysis of antibodies against FMDV of the presented genotype.

In addition, the prototype test system is based on a solid-phase competitive “sandwich” ELISA variant, however, although this form is one of the options for serological reactions, it is further from the neutralization reaction in terms of the ability to capture antigenic sites compared to the liquid-phase blocking indirect " sandwich" ELISA variant.

The prototype version assumes the presence in its composition 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 also has some limitations in use, namely, the working solution of the conjugate is not stable and quickly collapses. As part of the set, it is presented in the form of a 30-fold concentrate. In this state, in contrast to the lyophilized component, the shelf life is markedly reduced. It should also be noted that this test system does not provide for conjugate control, which does not allow obtaining more reliable data on optical density and antibody titer against FMDV.

In the prototype test system, a direct conjugate is used, which is convenient to use, however, this increases the likelihood of noise background phenomena, which can adversely affect the final results of a quantitative study of animal blood sera.

In the prototype test system, TMB dye, which has carcinogenic properties, is used to develop a color reaction.

Given the found limitations in the use of existing test systems, including the prototype, for accurate analysis of the humoral immunity of animals after inoculation with FMD vaccines containing inactivated FMDV strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2, relevant is the development of a serological test system for a liquid-phase blocking indirect "sandwich" ELISA variant for the detection of antibodies in animal blood sera, taking into account the above disadvantages.

Currently, the veterinary service of the Russian Federation does not have a modern domestic ELISA test system that would allow to determine with high reliability the titer of antibodies against FMDV strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2 in animal sera.

The aim of the invention is to create a test system based on a liquid phase blocking indirect "sandwich" ELISA variant for determining the antibody titer against FMDV strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2.

This goal is achieved by the fact that the proposed test system contains the following immunospecific components:

1) cultural inactivated lyophilized foot-and-mouth disease virus strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2;

2) sensitizing antibodies - lyophilized rabbit immunoglobulins G against foot-and-mouth disease virus strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2;

3) detector antibodies - lyophilized guinea pig immunoglobulins G against FMDV strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2;

4) control 1 - lyophilized positive blood serum of calves to the antigen of foot-and-mouth disease virus strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2, with a percentage of inhibition ≥75%;

5) control 2 - lyophilized positive blood serum of calves to the antigen of foot-and-mouth disease virus strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2, with a percentage of inhibition from 50% to 75%;

6) control 3 - lyophilized blood serum of calves, not containing antibodies to foot-and-mouth disease virus - negative control;

7) freeze-dried fetal bovine serum normal to block open binding sites at the bottom of the wells of the tablet;

8) anti-species conjugate - lyophilized rabbit immunoglobulins against guinea pig IgG, conjugated with horseradish peroxidase.

The proposed test system also contains non-specific components: carbonate-bicarbonate buffer (pH 9.5-9.6) for antigen dilution, 20-fold buffer solution concentrate, chromogenic substrate ABTS (2,2-azino-di-(3-ethylbenzoaminosulfonic acid), "stop" solution - 1% solution of sodium dodecyl sulfate.

The goal is achieved due to the fact that the liquid-phase blocking indirect "sandwich" ELISA variant is closest to the classical neutralization reaction in vivo, since all antigenic sites of the full virions of the FMDV strain O No. 2047 / Saudi Arabia / 2008 genotype O / ME-SA / PanAsia2 is free in the liquid phase, open to binding with rabbit and guinea pig specific sensitizing and detection antibodies, respectively. The reaction is based on the interaction of the studied animal blood serum and inactivated foot-and-mouth disease virus strain O No. 2047/Saudi Arabia/2008 of the O/ME-SA/PanAsia2 genotype 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 cattle blood serum, the resulting immune complexes are added from a round-bottom plate, incubated, and a reconstituted suspension of detection antibodies is 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 consists in the development of a modern, highly specific and sensitive test system designed to determine the titer of antibodies against foot-and-mouth disease virus strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2 using the liquid-phase blocking indirect "sandwich" option ELISA, due to the use of inactivated FMDV strain O No. 2047 / Saudi Arabia / 2008 genotype O / ME-SA / PanAsia2 and upon receipt of specific reaction components in the liquid phase.

The developed ELISA test system includes the following immunospecific components:

1. cultural inactivated lyophilized foot-and-mouth disease virus strain O No. 2047 / Saudi Arabia / 2008 genotype O / ME-SA / PanAsia2 - 1 bottle (0.5 cm ³ );

2. sensitizing lyophilized antibodies - rabbit immunoglobulins G against foot-and-mouth disease virus strain O No. 2047 / Saudi Arabia / 2008 genotype O / ME-SA / PanAsia2 - 1 bottle (0.5 cm ³ );

3. detector freeze-dried antibodies - guinea pig immunoglobulins G against foot-and-mouth disease virus strain O No. 2047 / Saudi Arabia / 2008 genotype O / ME-SA / PanAsia2 - 1 bottle (0.5 cm ³ );

4. control 1 - positive lyophilized blood serum of cattle to foot-and-mouth disease virus strain O No. 2047 / Saudi Arabia / 2008 genotype O / ME-SA / PanAsia2, with a percentage of inhibition ≥75% - 1 vial (0.2 cm ³ );

5. control 2 - positive lyophilized blood serum of cattle to foot-and-mouth disease virus strain O No. 2047 / Saudi Arabia / 2008 genotype O / ME-SA / PanAsia2, with a percentage of inhibition from 50% to 75% - 1 bottle (0.2 cm ³ );

6. control 3 - normal lyophilized blood serum of cattle, not containing antibodies to foot-and-mouth disease virus - 1 vial (0.2 cm ³ );

7. freeze-dried fetal bovine serum normal to block open binding sites at the bottom of the wells of the tablet - 2 vials (2.0 cm ³ each);

8. anti-species freeze-dried conjugate - rabbit immunoglobulins against guinea pig IgG, conjugated with horseradish peroxidase.

The proposed test system contains non-specific components:

9. carbonate-bicarbonate buffer solution (pH 9.5-9.6) for antigen dilution;

10. 20x buffer solution concentrate;

11. chromogenic substrate - dye ABTS (2,2-azino-di-(3-ethylbenzoaminosulfonic acid);

12. "stop" solution - 0.5% solution of sodium dodecyl sulfate.

The test system also includes 4 immunological 96-well flat-bottom polystyrene plates for ELISA and 1 polymer 96-well round-bottom plate for antigen and antibody binding in the liquid phase.

Unlike the prototype, the developed test system uses cultured inactivated FMDV strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2, which is presented as a lyophilisate. This fact makes it possible to detect precisely antibodies against FMDV of the specified strain with high sensitivity (more than 99%) and specificity (100%), which is more than 40% higher compared to the closest prototype. In addition, the lyophilized component remains stable for at least 3 years compared to the antigen in the native state, which is an advantage in using this test system. The activity of the obtained antigen of FMDV strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2 is 1:3500.

In contrast to the prototype, sensitizing and detection antibodies of rabbit and guinea pig are used, respectively, highly specific for the antigen of the foot-and-mouth disease virus strain O No. 2047 / Saudi Arabia / 2008 of the O / ME-SA / PanAsia2 genotype, since they were obtained using a highly purified antigen with a high activity. The rabbit and guinea pig immunoglobulins G against FMDV strain O No. 2047/Saudi Arabia/2008 of the O/ME-SA/PanAsia2 genotype specified in the ELISA test system are presented as a lyophilic component, which allows the antibodies to be in a stable state for at least 3 years from activity not less than 1:10000 and 1:4500, respectively.

In contrast to the prototype bovine blood serum, controls 1 and 2, were obtained against highly purified inactivated FMDV strain O No. 2047/Saudi Arabia/2008 of the O/ME-SA/PanAsia2 genotype, which makes it possible to achieve high specificity and sensitivity when monitoring the operation of the test system . These components are presented in the form of a lyophilisate, which makes it possible to store them for at least 3 years while maintaining high activity in ELISA. For control 1, the activity is at least 1:4000, for control 2 - at least 1:4000. This activity is high and convenient for obtaining a large number of controls in the working native state.

Unlike the prototype, purified 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 a freeze-dried form, which allows it to be stored in this state for at least 3 years.

Unlike the prototype version, the presented test system provides for the control of antigen and conjugate, which makes it possible to take into account background values and, thereby, obtain reliable absolute data on optical densities and the titer of antibodies against FMDV strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2.

Unlike the prototype, the proposed test system uses an indirect conjugate, which makes it possible to reduce the likelihood of noise background phenomena and obtain more reliable results for determining the antibody titer against FMDV strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2 in sera animal blood. The activity of the conjugate is 1:3000.

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. 2047/Saudi Arabia/2008 (genotype O/ME-SA/PanAsia2).

Fig. 3 - Electropherogram for the antigen of foot and mouth disease virus strain O No. 2047/Saudi Arabia/2008 (genotype O/ME-SA/PanAsia2) in 12% polyacrylamide gel. Note: 1-3: 50% sucrose fractions, 4-8: 40% sucrose fractions, 9-13: 30% sucrose fractions, 14-18: 20% sucrose fractions; target protein with a molecular weight of 25 kDa.

Fig. 4 - Phylogenetic affiliation of the FMDV isolate O/SAU/5/2008 / strain O No. 2047/Saudi Arabia/2008, genotype O/ME-SA/PanAsia2.

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

SEQ ID NO:1 represents the nucleotide sequence of the 1D gene of strain O No. 2047/Saudi Arabia/2008 of foot-and-mouth disease virus (genotype O/ME-SA/PanAsia2);

SEQ ID NO:2 represents the amino acid sequence of the VP1 protein of strain O No. 2047/Saudi Arabia/2008 of foot and mouth disease virus genotype O/ME-SA/PanAsia2.

The developed test system based on the liquid-phase blocking indirect "sandwich" ELISA variant is based on the specific blocking of the antigen of the foot-and-mouth disease virus strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2 by antibodies contained in the test serum sample in the liquid phase. The “test serum/antigen” mixture is transferred into the wells of the plate, previously immobilized with rabbit sensitizing antibodies against strain O No. 2047/Saudi Arabia/2008 of genotype O/ME-SA/PanAsia2. The presence of antibodies to foot-and-mouth disease virus strain O No. 2047 / Saudi Arabia / 2008 genotype O / ME-SA / PanAsia2 in the test sample will be expressed in the formation of an immune complex and a decrease in the amount of free antigen that binds sensitizing antibodies. The antigen fixed in the wells interacts with guinea pig detection antibodies, which are detected by reaction with an immunoperoxidase conjugate against guinea pig IgG and subsequent staining with 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. A schematic diagram of a liquid-phase blocking indirect "sandwich" ELISA variant is shown in Fig. 1.

Before starting work, the components are incubated for 30 minutes at a temperature of 18 to 23°C.

The lyophilized components of the test system are prepared. Before starting work, lyophilized preparations (antigen of foot and mouth disease virus strain O No. 2047/Saudi Arabia/2008 (genotype O/ME-SA/PanAsia2), conjugate, positive and negative controls, fetal calf blood serum) are restored to the required volume with distilled water.

Conduct the preparation of working solutions.

Solution No. 1 (carbonate-bicarbonate buffer solution - CBB). Prepare according to the standard method 100 ml of CBB solution (pH 9.5-9.6) for sensitization of the tablets.

Solution No. 2 (washing solution). 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. Prepare 2 liters of standard TBS buffer solution with the addition of 0.05% tween-20 (pH 7.4-7.6).

Solution No. 3 (buffer for recovery of antibodies and conjugate). 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 calf serum. To do this, 4.0 cm ³ of fetal calf serum, restored from a lyophilized preparation by adding 4.0 cm ^{3 of} distilled water to the contents of the vial with serum ^, should be added to 36 cm 3 of solution No. 2. Prepared immediately before use.

The stages of the liquid-phase blocking indirect "sandwich" ELISA variant to determine the titer of antibodies against FMDV strain O No. 2047/Saudi Arabia/2008 (genotype O/ME-SA/PanAsia2) are presented below.

1. Sensitization of tablets. A reconstituted suspension of sensitizing antibodies is prepared at a 1:10,000 dilution in solution No. 1 (carbonate-bicarbonate buffer solution). Into all wells of the immunological polystyrene 96-well plate for ELISA, 100 μl of a suspension of sensitizing antibodies are added, covered and incubated for 18-20 hours at a temperature of (2-8)°C.

2. Washing the wells of the tablet is carried out using a Washer of any brand, or manually with solution No. 2. The procedure is repeated twice.

3. Serological reaction 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: 4.0; 4.5; 5.0; 5.5; 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; 9.0: 9.5; 10.0; 10.5 log ₂ (1:16-1:1448) in solution #2. To do this, 0.047 cm ³ (47 μl) of buffer solution and 0.003 cm ³ (3 μl) of the sample are added to all wells of the polymer plate, with the exception of wells H1-6, which are left for antigen control (CA) and conjugate control (CC). In the wells H1-6 make 0.05 cm ³ (50 µl) buffer solution. The test and control samples are placed on the tablet according to table 1.

As control 1, positive blood serum of pigs against foot-and-mouth disease virus strain O No. 2047 / Saudi Arabia / 2008 (genotype O / ME-SA / PanAsia2) is used - the percentage of inhibition is 75-100%, control 2 is positive blood serum of pigs against foot-and-mouth disease virus strain O No. 2047/Saudi Arabia/2008 (genotype O/ME-SA/PanAsia2) - inhibition percentage 50-74%, control 3 - normal porcine serum - inhibition percentage less than 50%.

In all wells of the tablet add 0.05 cm ³ working dilution of the antigen in solution No. 2 in accordance with a dilution of 1:3500. After mixing the contents of the wells of the tablet using a shaker or light shaking by hand, the tablet is covered with a lid and incubated for 18-20 hours at a temperature of (2-8)°C.

4. Antigen capture. 0.05 cm ³ 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 60±5 min at a temperature of (37±1)°C.

5. Washing the wells of the tablet. Carry out as described above (p. 2). The procedure is repeated 4 times.

6. Introduction of detection antibodies. In all wells of the plate, except for the wells with QC (conjugate control), where 0.05 cm3 of solution No. 3 is added, 0.05 cm ³ of the working dilution of detection antibodies (1:4500) in solution No. 3 is added. The tablet is covered with a lid and incubated for 60±5 min at a temperature of (37±1)°C.

7. Washing the wells of the tablet. Carry out as described above (p. 2). The procedure is repeated 4 times.

8. Introduction of the conjugate. In all wells of the tablet contribute 0.05 cm ³ of the working dilution of the conjugate (1:3000) in solution No. 3. The tablet is covered with a lid and incubated for 60±5 min at a temperature of (37±1)°C.

9. Washing the wells of the tablet. Carry out as described above (p. 2). The procedure is repeated 4 times.

10. The manifestation of a color reaction. 0.05 cm ³ of the ABTS chromogenic substrate is added to all wells of the tablet, closed with a lid and incubated for 15-20 minutes at a temperature of (18-23)°C.

11. Stop the reaction. The reaction is stopped by adding to each well of the tablet 0.05 cm ³ 0.5% solution of sodium dodecyl sulfate.

12. Accounting for the results of ELISA. The results of the analysis are taken into account in an instrumental way. Immediately after stopping the reaction, measure the optical density (D) of the reaction products in each well at a wavelength of 410 nm using a spectrophotometer-reader for microplates with a vertical beam of light. The D values of the control and test samples are converted into the percentage of inhibition (PI, %) according to the formula:

PI (%)=(1-(D _serum -D _KK )/(D _KA -D _KK ))×100,

where D _serum is the average value of the optical density of a mixture of serum with inactivated FMDV strain O No. 2047/Saudi Arabia/2008 (genotype O/ME-SA/PanAsia2);

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

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

The results of the reaction are reliable if the data meet the following criteria:

- the difference between the values of D _KA and D _QC is not less than 0.4 optical units (r.u.);

- control 1 has a PI value>75%;

- control 2 has a PI value in the range of 50-74%;

- Control 3 has a PI<50%.

Samples showing a PI value of ≥50% are considered positive, and animals from which these blood serum samples are obtained are immune to FMD caused by the virus strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2. The value of PI<50% in the blood serum of the examined animals is negative, which indicates the absence of specific antibodies to foot-and-mouth disease caused by the virus strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2. 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. 2047/Saudi Arabia/2008.

As a result of the experiments, the conditions for setting up the reaction were optimized. The activity of detection antibodies, determined in an indirect ELISA, was 1:4500, anti-species conjugate - 1:3000; sensitizing antibodies - 1:10000, antigen of foot-and-mouth disease virus strain O No. 2047 / Saudi Arabia / 2008 (genotype O / ME-SA / PanAsia2) - 1:3500, control 1 - 1:4000, control 2 - 1:4000, control 3 - 1:3000. Using the developed test system, we studied animal blood serum samples with the determination of antibody titer and compared the results of the neutralization reaction in a sensitive continuous monolayer porcine kidney cell line (IB-RS-2) [2].

The developed version of the ELISA allows you to test simultaneously in the format of a 96-well plate 6 serum samples in the following dilutions, expressed in binary logarithm: 4.0; 4.5; 5.0; 5.5; 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; 9.0: 9.5; 10.0; 10.5 log ₂ (1:16-1:1448). The shelf life of the proposed test system is at least 24 months from the date of manufacture at a storage temperature of 4-8°C.

As a worker, inactivated FMDV strain O No. 2047/Saudi Arabia/2008 (genotype O/ME-SA/PanAsia2) was used.

Control of strain O No. 2047/Saudi Arabia/2008 of foot and mouth disease virus for specificity. Virus isolate, which served as a source for obtaining strain O No. 2047/Saudi Arabia/2008 of FMDV, was isolated from a calf in Saudi Arabia. Specificity was determined by RT-PCR followed by sequencing of the 1D gene corresponding to the VP1 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. 2047/Saudi Arabia/2008 was identified as belonging to the O/ME-SA/PanAsia2 genotype. This sequence is closest to the nucleotide sequence presented in GenBank under the number MT 443784.1 (isolate O/SAU/5/2008) [19]. The calculation was carried out in accordance with the Bayesian probability theory. Specificity was also confirmed in the reaction of complement fixation with strain-specific serum. The concentration of total viral protein in the studied antigen of foot and mouth disease virus strain O No. 2047 / Saudi Arabia / 2008 was 2.58 μg / ml, the concentration of the 146S component was 1.68 μg / ml, the 75S component was 0.77 μg / ml, 12S - 0 .13 µg/ml.

The possibility of using strain O No. 2047/Saudi Arabia/2008 of the FMD virus for control of antigenic and immunogenic activity, as well as for the manufacture of biological preparations for the diagnosis and specific prevention of FMD type O has been experimentally confirmed.

Morphological features

The FMD virus strain O No. 2047/Saudi Arabia/2008 belongs to the family Picornaviridae, genus Aphthovirus, serotype O, genotype O/ME-SA/PanAsia2 and has morphological features characteristic of the FMD pathogen: the virion form is icosahedral, 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, strain O No. 2047/Saudi Arabia/2008 of the virus is stably neutralized by homologous antiserum. The virus does not show hemagglutinating activity. In ill animals, antibodies are formed in the blood serum, which are detected in the enzyme immunoassay (ELISA) and neutralization tests.

During hyperimmunization of guinea pigs, a concentrated antigen from an inactivated foot-and-mouth disease virus of strain O No. 2047 / Saudi Arabia / 2008 induces the formation of virus-specific antibodies detected in RSK at a dilution of 1:3500.

Antigenic relatedness of strain O No. 2047/Saudi Arabia/2008 of foot-and-mouth disease virus with existing industrial strains of foot-and-mouth disease virus serotype O was studied by serological method in the cross-reaction of microneutralization (RMN). Strain O No. 2047/Saudi Arabia/2008 is antigenically different from production strains O1/Manisa/1993, O No. 1715/Taiwan/3/97, O No. 2147/Primorsky/2012, O No. 2212/Primorsky/2014, O No. 2311/ Zabaikalsky/2016, O No. 2344/Mongolia/2017.

The results of studies in the RMN are presented in table 2. Antigenic relationship (r ₁ ) amounted to O1/Manisa/1993 - 0.05; O No. 1715/Taiwan/3/97 - 0.09; O No. 2147 / Primorsky / 2012 - 0.12; O No. 2212 / Primorsky / 2014 - 0.09; O No. 2311 / Zabaikalsky / 2016 - 0.11; O No. 2344 / Mongolia / 2017 - 0.11. 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. 2047/Saudi Arabia/2008 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 14-20 hours of incubation of the virus in these cell cultures accumulates from 6.50 to 8.50 lg TCD ₅₀ /cm ³ .

Genotaxonomic characteristics.

Strain O No. 2047/Saudi Arabia/2008 of type O foot-and-mouth disease virus is an RNA-containing virus.

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.

For this virus, with a sedimentation constant of complete viral particles 146 S, a diffusion constant of 1.41 cm ² /s, the molecular weight is 8.08 × 10 ⁶ D. The size of the virion is 23-25 nm, the mass is 8.4 × 10 ^-18 g.

Resilience to external factors.

Strain O No. 2047/Saudi Arabia/2008 is resistant to ether, chloroform, freon, acetone and other organic solvents and detergents. Most stable at pH 7.5-7.8. 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 inactivated foot and mouth disease virus strain O No. 2047 / Saudi Arabia / 2008 genotype O / ME-SA / PanAsia2 guinea pigs, rabbits, pigs, sheep, cattle 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 cultivated 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 inactivated FMDV strain O No. 2047/Saudi Arabia/2008.

To obtain inactivated foot and mouth disease virus strain O No. 2047 / Saudi Arabia / 2008 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 suspension containing the FMDV antigen was clarified for 30 min. at 6000 rpm and 4°C in a Beckman J2-21 centrifuge (Beckman Coulter, USA) or equivalent. The supernatant liquid was taken and polyethylene glycol with a molecular weight of 6000 D (PEG-6000) was added to it to a final concentration of 8% and sodium chloride (dry) to a final concentration of 0.85%, intensively mixed and kept at a temperature of 4±2°C for 18-24 hours. The virus suspension was precipitated at 6000 rpm for 60 min., the precipitate was dissolved in 1/15 M PBS solution, concentrating 200 times (1/200 of the original volume).

50% trichloromethane was added to the resulting precipitate, vigorously stirred, and fractionated using a centrifuge for 30 min. at 3000 rpm. Selected the upper water fraction containing the antigen of FMDV strain O No. 2047/Saudi Arabia/2008. A 100 μl sample was taken for subsequent electrophoretic analysis in a 12% polyacrylamide gel.

At the next stage, the 146S component of foot-and-mouth disease virus strain O No. 2047/Saudi Arabia/2008 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 nonylphenoxypolyethoxylethanol 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%. Treated with nonylphenoxypolyethoxyethanol antigen of foot-and-mouth disease virus strain O No. 2047/Saudi Arabia/2008, 15 ml were poured into centrifuge tubes, then sucrose solutions were sequentially layered, starting with 10% and ending with 50% solution. The tubes were placed in metal centrifuge cups and after careful balancing were centrifuged at a speed of 25,000 rpm and a temperature of 4±2°C for 4 hours. Fractionation of the sucrose gradient was performed using a peristaltic pump, taking fractions of 1 ml into separate tubes. The beginning and end of the peak of the 146S component were determined from the spectral profile by combining the fractions within this range.

When using a peristaltic pump, the sucrose gradient tube was first visually evaluated. The opalescent layer containing the 146S component is located in approximately 30% sucrose layer. In this case, the last upper fraction of the 50% layer (1.0 ml), all fractions of the 30% layer (5.0 ml) and the lower fraction of the 20% layer of sucrose (1.0 ml) were taken. The total volume is approximately 7.0 ml. The 146S component was then reprecipitated by ultracentrifugation for 4 hours at 25,000 rpm. and a temperature of 4±2°C and resuspendable sediment in 1/15 M phosphate-buffered saline.

The results of a spectrometric study of the obtained fractions of the antigen of foot-and-mouth disease virus strain O No. 2047/Saudi Arabia/2008 in the form of an antigenic profile are shown in Fig. 2. The analysis was carried out for 20 fractions, peaks were observed for fractions 5-10 with maximum values for 7 and 8 peaks. Electrophoresis of the obtained fractions was carried out in 12% polyacrylamide gel under denaturing conditions for 18 fractions, in which the 146S component of the FMD virus of this strain can be found (Fig. 3). In FIG. 3 shows that slots No. 1-3 are fractions with 50% sucrose, Nos. 4-8 are fractions with 40% sucrose, Nos. 9-13 are fractions with 30% sucrose, Nos. 14-18 are fractions with 20% sucrose.

Example 2. Obtaining hyperimmune rabbit blood serum (sensitizing antibodies) against strain O No. 2047 / Saudi Arabia / 2008.

To obtain specific blood sera against foot-and-mouth disease virus strain O No. 2047 / Saudi Arabia / 2008, clinically healthy rabbits of average fatness weighing 2.5-3.0 kg were used.

For hyperimmunization of animals, concentrated purified inactivated FMDV strain

O No. 2047/Saudi Arabia/2008 prepared as described in Example 1, emulsified using Montanide ISA-70 VG oil adjuvant (adjuvant/antigen ratio = 70/30 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 blood serum of guinea pigs (detector antibodies) against FMDV strain O No. 2047/Saudi Arabia/2008.

For hyperimmunization of guinea pigs, an emulsion of a purified preparation of a concentrated inactivated foot-and-mouth disease virus of strain O No. 2047 / Saudi Arabia / 2008, 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 foot-and-mouth disease virus strain O No. 2047 / Saudi Arabia / 2008, 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, 1:9000, 1:10000, 1:12000. The dilutions of the remaining components were constant: for antigen - 1:3500, for detection antibodies - 1:4500, conjugate - 1:3000. Serums specific to certain strains of FMDV with an activity of 1:256 were used as control sera for analysis. It follows from Table 3 that a 1:10,000 dilution of the sensitizing antibodies achieved a reliable result (1:256 or 8 log ₂ /ml). At lower dilutions of antibodies, high background values were observed, which did not allow obtaining a correct result. 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: 1:3000, 1:4000, 1:4500, 1:5000. The dilutions of the remaining components were constant: for antigen - 1:3500, for sensitizing antibodies - 1:10000, conjugate - 1:3000. 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 dilutions of antibodies, high background values were observed, which did not allow obtaining a correct result. 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, 1:3500, 1:4000. The dilutions of the remaining components were constant: for detection antibodies - 1:4500, for sensitizing antibodies - 1:10,000, conjugate - 1:3000. 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 dilutions, high background values were observed, which did not allow obtaining a correct result. 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 FMDV antigen strain O No. 2047 / Saudi Arabia / 2008, sensitizing and detection antibodies were 1:3500, 1:10000, 1:4500, respectively.

Example 5. The use of a liquid-phase blocking indirect "sandwich" ELISA test system to determine the antibody titer against the antigen of foot-and-mouth disease virus strain O No. 2047 / Saudi Arabia / 2008.

We studied 12 blood sera of animals obtained after immunization with inactivated adsorbed FMD vaccines containing the antigen of FMDV strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2. Antibody titer values were determined using the developed ELISA test system, and serum data were studied in a neutralization reaction in a sensitive continuous monolayer cell line IB-RS-2 recommended by 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 ELISA test system (the proposed invention) and the classical method (RN) is high (approximately 100%).

Example 6. Determination of the diagnostic parameters of the developed test system for determining the antibody titer in the blood sera of animals against foot-and-mouth disease virus strain O No. 2047/Saudi Arabia/2008 using a liquid-phase blocking indirect "sandwich" ELISA variant.

To study the presented test system (the proposed invention) to determine the titer of antibodies against FMDV strain O No. 2047/Saudi Arabia/2008, standard diagnostic indicators were studied. To determine the sensitivity of the proposed test system, 625 animal sera were analyzed, which were known to be positive according to the neutralization test in a sensitive transplantable monolayer porcine kidney cell line IB-RS-2. The antibody titer for these blood sera was in the range of 1:32-1:1448. The ELISA was performed as described above. The developed test system (the proposed invention) determined that out of 625 blood serum samples, 622 were identified as positive, and 3 as negative (titer 1:16-1:24). To study the specificity of the method, 148 negative animal sera were tested. As a result of the study using ELISA (the present invention), it was determined that out of 148 negative samples, 148 were identified as negative. Using the above statistical methods of analysis, it was determined that the diagnostic sensitivity (DSe) was 99.52% (in the 95% confidence interval: 98.64-99.90%), the diagnostic specificity (DSp) was 100% (in 95% -nom confidence interval: 97.54-100.0%), k-test - 0.988; positive predictive value (PPV) - 100%, negative predictive value (NPV) - 99.01%) (95% confidence interval: 94.10-99.35%), overall accuracy (DAc) - 99.61 % (in 95% confidence interval: 98.87-99.92%) (Table 7).

Thus, the proposed "Test system based on liquid-phase blocking indirect "sandwich" ELISA for determining the titer of antibodies against FMDV strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2 in animal sera" is specific and sensitive, allowing you to quickly analyze animal blood sera within 4-5 hours with a quantitative determination of the titer of antibodies against FMDV strain O No. 2047 / Saudi Arabia / 2008 genotype O / ME-SA / PanAsia2.

Sources of information taken into account when compiling the description of the invention to the application for the grant of a patent of the Russian Federation for the invention "Test system based on a liquid-phase blocking indirect" sandwich "ELISA variant for determining the titer of antibodies against FMDV strain O No. 2047 / Saudi Arabia / 2008 genotype O/ME-SA/PanAsia2 in animal sera":

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2. OIE. Manual of diagnostic tests and vaccines for terrestrial animals. - Paris, 2018. - Chapter 2.1.8.

3. 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.

4. Anon Global Strategy for the control of foot-and-mouth disease. - In, Bangkok, Thailand, - 2012. - P. 27-29.

5. Dynamics of development of anti-foot-and-mouth humoral immunity in cattle immunized with trivalent adsorbed vaccine types A, O, Asia-1 / D.V. Mikhalishin, T.N. Lezova // Proceedings of the Federal Center for Animal Health. - Vladimir, 2007. - T. 5. - S. 75-82.

6. Wong C.L., Yong C.Y., Ong H.K., Ho K.L., Tan W.S. Advances in the Diagnosis of Foot-and-Mouth Disease // Front Vet Sci. - 2020 Aug 21. - V.7. - P. 477.

7. Curry S., Abu-Ghazaleh R., Blakemore W., Fry E., Jackson T., King A., Lea S., Logan D., Newman J., Stuart D. Crystallization and preliminary X-ray analysis of three serotypes of foot-and-mouth disease virus // J. Mol Biol. - 1992. - V. 228(4). - P. 1263-1268.

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14. Cao Y., Lu Z., Sun J., Bai X., Sun P., Bao H., Chen Y., Guo J., Li D., Liu X., Liu Z. Synthesis of empty capsid- like particles of Asia I foot-and-mouth disease virus in insect cells and their immunogenicity in guinea pigs // Vet. microbiol. - 2009. - V. 137(1-2). - P. 10-17.

15. Hamblin C, Barnett I.T., Hedger R.S. A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. I. Development and method of ELISA // J. Immunol. methods. - 1986. - V. 93(1). - P. 115-121.

16. Grubman M.J., Moraes M.P., Diaz-San Segundo F., Pena L., de los Santos T. Evading the host immune response: how foot-and-mouth disease virus has become an effective pathogen // FEMS Immunol Med Microbiol. - 2008. - V. 53(1). - P. 8-17.

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18. Diagnostic assays developed for the control of foot-and-mouth disease in India / Sharma G.K, Mahajan S, Matura R, Subramaniam S, Ranjan R, Biswal J, Rout M, Mohapatra J.K, Dash B.B, Sanyal A, Pattnaik B Diagnostic assays developed for the control of foot-and-mouth disease in India // World J Virology. - 2015. - V. 4(3). - P. 295-302.

19 GenBank. Foot-and-mouth disease virus - type О isolate SAU/5/2008 VPI gene, partial cds/ URL: https://www.ncbi.nlm.nih.gov/nuccore/2050239519 (Date of access: 01/05/2022).

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Sequence listing

<110> Federal State Budgetary Institution "Federal Center

Animal Health (FGBI ARRIAH) Federal Governmental Budgetary

Institution "Federal Center for Animal Health" (FGBI "ARRIAH")

<120> Test system based on liquid-phase blocking indirect

"sandwich"-variant of ELISA to determine the titer of antibodies against foot-and-mouth disease virus

strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2

in blood sera animals

<160> 2

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<213> foot-and-mouth disease virus

<400> 1

acccgcca ccggggaatc agcagaccct gtcacaacca ccgttgagaa ctacggtggc 60

gagacacaag tacagcggcg ttaccacacc gacgtcggct tcttaatgga caggttcgtg 120

cagatcaagc ctgtgggccc cacacatgtc attgacctca tgcagacaca ccaacacggg 180

ctggtgggcg ccatgttgcg cgcggccacc tactactttt ctgatcttga gattgtggtg 240

aaccacacgg gtaacctaac gtgggtaccc aatggagcac ccgaggcagc actgcaaaac 300

acgagcaacc ccactgctta ccacaaagcg ccgttcacga ggcttgcgct cccctacacc 360

gcgccacacc gcgtgctggc aactgtgtac agtgggacga gcaagtactc cgcacctcaa 420

aaccggcgag gtgacttggg tcctctcgcg gcgagactcg ctgcacagct ccctgcctcc 480

ttcaacttcg gtgcaattcg ggccacggag atctgcgaac tccttgtgcg catgaagcgc 540

gccgagctct actgccccag gccactgttg gcggtggagg tgtcgtcgca agacagacac 600

aagcagaaaa tcattgcccc tgcaaaacaa ctcctg 636

<210> 2

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<212> PRT

<213> foot-and-mouth disease virus

<400> 2

1 Thr Thr Ser Thr Gly Glu Ser Ala Asp Pro Val Thr Ala 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 Thr 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 Ser Cys Lys 135

136 Tyr Gly Glu Ser His Thr 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 Asp Glu Ala Arg His Lys Gln Lys Ile Val Ala Pro Val Lys 210

211 Gln Leu Leu 213

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Claims (4)

1. Test system based on liquid-phase blocking indirect "sandwich" ELISA for determining the titer of antibodies against FMDV strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2 in animal blood sera, containing - specific components: cultural inactivated lyophilized foot-and-mouth disease virus strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2; sensitizing antibodies - freeze-dried rabbit immunoglobulins G against FMDV strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2; detector antibodies - lyophilized guinea pig immunoglobulins G against FMDV strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2; control 1 - freeze-dried positive blood serum of calves to the antigen of foot-and-mouth disease virus strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2 with a percentage of inhibition ≥75%; control 2 - freeze-dried positive blood serum of calves to the antigen of foot-and-mouth disease virus strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2 with a percentage of inhibition from 50 to 75%; control 3 - lyophilized blood serum of calves that does not contain antibodies to foot-and-mouth disease virus - negative control; freeze-dried fetal bovine serum normal to block open binding sites; anti-species conjugate - lyophilized rabbit immunoglobulins against guinea pig IgG, conjugated with horseradish peroxidase; - non-specific components: carbonate-bicarbonate buffer pH 9.5-9.6 for antigen dilution; 20x buffer solution concentrate; chromogenic substrate ABTS - 2,2-azino-di-3-ethylbenzoaminosulfonic acid; "stop" solution - 1% solution of sodium dodecyl sulfate, characterized in that it contains cultured inactivated foot and mouth disease virus strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2 as an antigen of the FMD virus and to determine the percentage of inhibition a modified formula is used: PI(%)=(1-(D _serum -D _CC )/(D _KA -D _CC ))×100, while the antibody titer is taken as the highest dilution of the studied blood serum, for which the percentage of inhibition is ≥ 50%. 2. The test system according to claim 1, which is specific and sensitive, allowing you to quickly analyze animal blood sera within 4-5 hours to determine the level of antibodies against FMDV strain O No. 2047 / Saudi Arabia / 2008 genotype O / ME-SA /PanAsia2 and determine their titer in the range of 4.0-10.5 log ₂ (1:16-1:1448).

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