RU2843305C1 - Enzyme immunoassay system for assessing antigen and immunogenic activity of foot-and-mouth disease vaccine based on foot-and-mouth disease virus strain sat-2/xiv/2023 - Google Patents

Abstract

FIELD: veterinary virology; biotechnology.

SUBSTANCE: described is an enzyme immunoassay for assessing antigen and immunogenic activity of foot-and-mouth disease vaccine based on foot-and-mouth disease virus strain SAT-2/XIV/2023 containing immunospecific reagents and non-specific reagents.

EFFECT: developing the sensitive and specific immunoenzymometric test system of the foot-and-mouth disease virus strain SAT-2/XIV/2023 for evaluating antigen and immunogenic activity of foot-and-mouth disease vaccine.

2 cl, 5 dwg, 4 tbl, 5 ex

Description

The invention relates to the field of veterinary virology and biotechnology, in particular, to the creation of an enzyme immunoassay test system for assessing the antigenic and immunogenic activity of an anti-foot-and-mouth disease vaccine based on the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus.

Foot-and-mouth disease is an acute, highly contagious vesicular disease of domestic and wild cloven-hoofed ruminants caused by an RNA-containing aphthovirus of the Picornaviridae family and characterized by fever, aphthous lesions of the mucous and epithelial tissues of animals, and other symptoms of infection. There are seven classified serotypes of foot-and-mouth disease: A, O, C, SAT 1, SAT 2, SAT 3, and Asia 1. Foot-and-mouth disease causes significant economic losses due to its negative impact on livestock production and is an obstacle to regional and international trade in animals and animal products. According to estimates by the World Organization for Animal Health (WOAH, founded as the OIE), the disease circulates in 77% of the world's livestock in Africa, the Middle East, and Asia, as well as in a limited area of South America [1, 2]. Therefore, there is a risk of infection being introduced into foot-and-mouth disease-free countries without vaccination.

Foot-and-mouth disease control involves timely preventive measures, such as effective epidemiological disease control; vaccination of domestic and farm animals against foot-and-mouth disease; surveillance of the movement of wild ungulates in border areas at risk of pathogen penetration from countries with unfavorable foot-and-mouth disease; monitoring studies to assess the immune status of agricultural and domestic livestock, including retrospective diagnostics of foot-and-mouth disease. Since the virus of different serotypes practically does not cause cross-immunity and when using vaccines based on pathogen strains other than field ones, even within one serotype, protection may be weak, when developing vaccines, careful selection of strains is necessary to ensure the maximum possible protection of livestock from foot-and-mouth disease in a particular region [1, 2, 3].

Outbreaks of foot-and-mouth disease of serotype SAT 2 are periodically recorded on the territory of the African continent. Representatives of the foot-and-mouth disease virus of serotype SAT 2 are characterized by high genetic variability and are divided into 14 topotypes (I - XIV). Such genetic and antigen diversity leads to problems in the specific prevention of foot-and-mouth disease when using culture-based inactivated foot-and-mouth disease vaccines, and also complicates the strain-specific diagnostics of the isolated isolates of the foot-and-mouth disease virus. As a result, there is a need to create new diagnostic tools and specific immunoprophylaxis against foot-and-mouth disease of serotype SAT 2 [1, 2].

Currently, the epizootic situation on the African continent is mainly associated with representatives of the causative agent of this disease serotype SAT 2 topotype VII (genotype SAT2/VII), but in 2022, the rapid spread of foot-and-mouth disease virus serotype SAT 2 topotype XIV (genotype SAT2/XIV) began, as a result of which it went beyond endemic territories. Outbreaks of foot-and-mouth disease genotype SAT2/XIV were registered in 2023 in some countries of the Eurasian continent, including Iraq, Jordan, and Turkey (Fig. 1).

The FMD virus serotype SAT 2 circulating in Iraq in 2023 belongs to topotype XIV, which is closely related to the virus isolates from Ethiopia in 2022 (SAT2/ETH/3/22 and SAT2/ETH/2/22). Both viruses were isolated from infected cattle in March 2022. The virus currently circulating in Jordan is also reported to be closely related to this Iraqi virus. The spread of exotic FMD viruses to large susceptible cattle and buffalo populations in Iraq threatens large animal populations in Iran, Turkey and other Middle Eastern countries and is of great concern [4].

Close cooperation with the CIS and Middle East countries, trade and economic relations of the Russian Federation with the countries of the African continent, where preventive vaccination of livestock against foot-and-mouth disease of serotype SAT 2 is not carried out, as a result of which there is a serious risk of introducing the foot-and-mouth disease virus of this serotype into the territory of the Russian Federation. In this regard, the production of vaccines against foot-and-mouth disease of serotype SAT 2, including genotype SAT2/XIV, the development and implementation of diagnostic test systems for assessing the effectiveness of vaccination and the intensity of immunity against foot-and-mouth disease of genotype SAT2/XIV in agricultural and domestic cloven-hoofed animals, as well as for the detection of post-infection anti-foot-and-mouth disease antibodies in the blood serum of unvaccinated animals are becoming increasingly important.

The FGBU "ARRIAH" has developed and successfully tested a mono- and polyvalent anti-foot-and-mouth disease vaccine based on the antigen of the "SAT-2/XIV/2023" strain of the foot-and-mouth disease virus [5]. This strain was deposited in the All-Russian State Collection of Exotic Types of Foot-and-Mouth Disease Virus and Other Animal Pathogens (GKShM) of the FGBU "ARRIAH" in 2023 under the registration number: No. 489 - dep / 23-39 - ГКШМ FGBU "ARRIAH". A patent was also obtained for the "SAT-2/XIV/2023" strain of the SAT-2/XIV genotype of the foot-and-mouth disease virus [6].

The strain "SAT-2/XIV/2023" of foot-and-mouth disease virus was obtained by adaptation to reproduction in the primary trypsinized monolayer cell line of pig kidney SP, in continuous cell cultures of BNK-21/SUSP/ARRIAH, IB-RS-2, PSGK-30, as well as in the body of cattle and pigs of the foot-and-mouth disease virus isolate, isolated at the Federal State Budgetary Institution "ARRIAH" from pathological material collected from cattle in the Hashemite Kingdom of Jordan in August 2023. The strain was proposed for the manufacture of vaccines and diagnostic tools.

The vaccine efficacy, immune background and intensity of anti-FMD immunity in susceptible animals are assessed by the level of specific virus-neutralizing (protective) antibodies in the blood serum of animals after vaccination or infection. The foot-and-mouth disease virion is non-enveloped and is a capsid surrounding the RNA genome. The capsid, which has an icosahedron structure with a sedimentation coefficient of 146S, is formed from 60 copies of the protomeric subunit consisting of four structural proteins VP ₁ -VP ₄ . The surface proteins VP ₁ -VP ₃ carry epitopes responsible for the serotype specificity of the foot-and-mouth disease virus and the production of virus-neutralizing antibodies, while the internal protein VP ₄ is more conservative in different serotypes of the pathogen and antibodies against VP ₄ epitopes do not provide protection against infection [7, 8].

In laboratory diagnostics, two main methods are used to study blood serum samples for the presence of antibodies to the structural proteins of the foot-and-mouth disease virus: the virus neutralization reaction (VNR) and enzyme-linked immunosorbent assay (ELISA) [9, 10].

The foot-and-mouth disease virus neutralization test is rightfully considered the gold standard, as it allows for the direct detection of virus-neutralizing antibodies in the blood serum of vaccinated or recovered animals, thereby determining the level of protection against a certain serotype of foot-and-mouth disease. However, there are a number of factors that limit or complicate the use of the test in laboratories. These include the need to work with live foot-and-mouth disease virus, which requires a laboratory biosafety level of at least III, duration of time (at least 72 hours), the need to use _CO2 to improve the growth properties of cell culture, the dependence of the test results on the state of the cell culture and the dose of the virus, etc.

The enzyme immunoassay, based on the use of an inactivated whole-virus or recombinant antigen in the reaction, covers the entire spectrum of virus-specific antibodies, primarily virus-neutralizing ones, which is due to the structure of the foot-and-mouth disease virion. The method is less time-consuming than RN and easier to set up. All this allows the use of ELISA for the diagnosis of foot-and-mouth disease in laboratory practice along with RN as an alternative or confirmatory method.

The proposed enzyme immunoassay test system for assessing the antigenic and immunogenic activity of the foot-and-mouth disease vaccine based on the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus of the SAT2/XIV genotype is based on the reaction of the liquid-phase blocking variant of ELISA.

This version of ELISA was first proposed by the staff of the Pirbright Institute, UK, which holds the title of World Reference Laboratory, Hamblin, et al., 1986, and recommended by WOAH as one of the main methods of retrospective diagnostics of foot-and-mouth disease: for assessing the intensity of immunity, for monitoring the immunogenicity of foot-and-mouth disease vaccines, for monitoring studies on foot-and-mouth disease [10, 11, 12, 13]. This version of ELISA was adapted and optimized for the conditions of the FGBU "ARRIAH".

The liquid-phase blocking variant for determining antibodies to structural proteins of the foot-and-mouth disease virus is a type of competitive ELISA and is distinguished by the presence of a preliminary stage of interaction of the foot-and-mouth disease virus antigen with the serum being tested ("liquid phase"), during which immunodominant epitopes in the amino acid sequence of the foot-and-mouth disease virus antigen are blocked by specific immunoglobulins. The remaining stages of the reaction are generally similar to other ELISA variants. Competing antibodies are homologous strain-specific polyclonal antibodies in the blood serum of a guinea pig (detector antibodies), binding to free antigenic determinants.

The undeniable advantage of this ELISA variant is the fact that the formation of the "antigen-antibody" complex occurs in the liquid phase, i.e. under conditions as close to natural as possible. In this case, there is no deformation of the virion and the antigen sites do not change, which makes it similar to the reaction of viral neutralization in cell culture or chicken embryos.

After the "liquid phase" stage, the further reaction takes place on a solid carrier ("solid phase"): a mixture of the serum sample and antigen under study is transferred to the wells of a 96-well plate, previously sensitized with specific capture antibodies (rabbit blood serum). Free unbound antigen interacts with specific capture antibodies and detector antibodies (guinea pig blood serum) to form a new immune complex, which is detected by secondary antibodies to guinea pig immunoglobulin G conjugated with horseradish peroxidase. This procedure is necessary to visualize the reaction during the process of staining the contents of the wells of the plate with a chromogenic substrate. In this case, the less specific antibodies the sample contains, the more intense the color.

To date, using liquid-phase blocking ELISA with the features of the reaction and a certain composition of the reaction components, the following have been developed: "A test system based on a liquid-phase blocking indirect "sandwich" ELISA variant for determining the titer of antibodies against foot-and-mouth disease virus strain "A No. 2269/VNIIZh/2015 genotype A/ASIA/G-VII" in the blood serum of animals after immunization", "A test system based on 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 the blood serum of animals after immunization" and "A method for 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 foot-and-mouth disease virus after vaccination using a liquid-phase “sandwich” ELISA variant” [14-16].

Currently, there is only one reliably known kit for retrospective diagnostics of foot-and-mouth disease presented on the world market, a test system that allows detection of antibodies to structural proteins of the foot-and-mouth disease virus serotype SAT 2 in ELISA. This is the Solid-phase competitive ELISA (SPCE) kit for antibodies specific to FMDV serotype SAT 2 (prototype) developed jointly by the Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna Brescia (IZSLER), Italy, and The Pirbright Institute, Pirbright, UK, manufactured by IZSLER. Information about this test system first appeared in 2014 in the materials of the Open session of the standing Technical and Research Committees of the EuFMD. Cavtat (Croatia), 29-31 October 2014) [17]. The kit for detecting antiviral antibodies in serum or plasma samples from infected or vaccinated susceptible animals against foot-and-mouth disease is based on a solid-phase competitive ELISA variant using virus-neutralizing monoclonal antibodies specific to the SAT 2 serotype (prototype).

The set includes:

- ELISA microplates, pre-sensitized with inactivated foot-and-mouth disease virus serotype SAT 2, captured by homologous monoclonal antibodies (MAb) – 5 plates;

- conjugate of MAb obtained against foot-and-mouth disease virus serotype SAT-2 with horseradish peroxidase, 10x (2.5 ml) - 1 vial;

- positive control serum to foot-and-mouth disease virus serotype SAT-2 (300 µl) – 1 bottle;

- negative control (2 ml) – 1 bottle;

- phosphate-buffered washing solution with the addition of TWEEN detergent, 10x concentrate (250 ml) – 1 bottle;

- ELISA buffer for dilution of serum and conjugate (120 ml), ready to use – 1 bottle;

- TMB substrate/chromogen solution (30 ml), ready to use – 1 bottle;

- stop solution 0.6N H2SO4 (30 ml), ready to use – 1 bottle.

During the screening test, the blood serum sample to be tested is diluted 1:10 with ELISA buffer in the wells of an ELISA microplate, where specific immunoglobulins interact with the foot-and-mouth disease virus antigen if present in the blood serum sample. When the MAb-HRP conjugate is added to the wells of the plate at a subsequent stage, its reaction with the homologous antigen will be inhibited by antibodies of the positive sera that have previously bound to the virus; in the case of negative sera, the conjugated MAbs freely bind to free antigenic determinants. The colorimetric reaction after the addition of the TMB substrate/chromogen at the stage of visualization of the test results develops if the serum to be tested is negative and the MAbs have formed an immune complex with the foot-and-mouth disease virus.

The results of the analysis are taken into account at a wavelength of 450 nm, recording the OD values in each well of the plate using a spectrophotometer (reader) for microplates with a vertical light beam. The OD values of the control and test samples are converted into the value of the percentage of inhibition (PI) using the formula:

PI = 100 – (serum _OD / _control OD *) × 100%;

where, *OD control is the average optical density value for 4 wells with negative control.

The test must meet the following validation criteria:

- OD values in negative control wells should be ≥1.0;

- positive control demonstrates ≥80% inhibition at 1:10 dilution and >50% inhibition at the second 1:30 dilution.

It is quite conditional to call the SPCE kit for antibodies specific to FMDV serotype SAT 2 manufactured by IZSLER (SAT2-IZSLER) a prototype of the proposed test system for assessing the antigenic and immunogenic activity of the foot-and-mouth disease vaccine based on the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus, as the test systems are very different from each other. Although both test systems are designed to detect antibodies to structural proteins of the foot-and-mouth disease virus of serotype SAT 2 in ELISA, the proposed invention is a strain-specific polyclonal test system based on a liquid-phase blocking version of ELISA, while the IZSLER kit is a serotype-specific monoclonal competitive test system.

The use of monoclonal antibodies in diagnostic enzyme immunoassay test systems for serotyping specific antibodies to the foot-and-mouth disease virus is intended to create universality of analysis within a specific foot-and-mouth disease serotype – the ability to detect antibodies produced to different strains of the foot-and-mouth disease virus of the same serotype with the same sensitivity in the studied serum or plasma samples, i.e. the test system must have broad serotype specificity.

However, in the case of the SAT2-IZSLER test system, pronounced genotype specificity was observed. When studying monospecific blood serum samples of cattle or pigs to the foot-and-mouth disease virus of the strains "SAT2/ERI/1998" (genotype SAT2/VII), "SAT2/LIB/39/2012" (genotype SAT2/VII), "SAT-2/XIV/2023" (genotype SAT2/XIV) of the foot-and-mouth disease virus in the SAT2-IZSLER test system, the greatest number of positive results were obtained for serum samples to the SAT2/VII genotype, while antibodies to the SAT2/XIV genotype were detected with less efficiency than in the presented invention. This indicates that the use of the SAT2-IZSLER kit to study the antigenic and immunogenic activity of the foot-and-mouth disease vaccine containing the antigen of the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus does not allow an objective and reliable assessment of the vaccine's effectiveness using the enzyme immunoassay method. Based on this, it is necessary to develop a sensitive, specific enzyme immunoassay test system to assess the antigenic and immunogenic activity of the foot-and-mouth disease vaccine based on the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus. Thus, the enzyme immunoassay test system for assessing the antigenic and immunogenic activity of the foot-and-mouth disease vaccine based on the SAT-2/XIV/2023 foot-and-mouth disease virus strain, which has a 100% degree of homology with the SAT-2/XIV/2023 foot-and-mouth disease virus vaccine strain and has high diagnostic indicators, is an indispensable, currently uncontested tool for assessing the quality of the vaccine in ELISA.

Taking into account the specified limitations in the use of existing test systems, including the closest prototype, for the accurate determination of the antigenic and immunogenic activity of foot-and-mouth disease vaccines containing the foot-and-mouth disease virus of the SAT2/XIV genotype, it is relevant to develop an enzyme immunoassay test system for assessing the antigenic and immunogenic activity of the foot-and-mouth disease vaccine based on the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus.

Currently, the veterinary service of the Russian Federation does not have a modern domestic ELISA test system that would allow for a highly reliable assessment of the antigenic and immunogenic activity of foot-and-mouth disease vaccines based on the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus.

The aim of the invention is to create an enzyme immunoassay test system for assessing the antigenic and immunogenic activity of an anti-foot-and-mouth disease vaccine based on the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus.

The uniqueness of the proposed test system for determining antibodies to structural proteins of the foot-and-mouth disease virus of the SAT2/XIV genotype consists in the use of the following specific reaction components in obtaining the following: foot-and-mouth disease virus antigen, capture and detector antibodies, positive and weakly positive control sera, and the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus. This test system has a 100% degree of homology with vaccine products that include the inactivated antigen of the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus, which allows for the most reliable and effective assessment of the antigenic and immunogenic properties of the foot-and-mouth disease vaccine after its administration to animals. The level of post-vaccination immunity is determined by the amount of specific antibodies in the blood serum of immunized cattle, expressed through the percentage of inhibition (PI) in accordance with 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 foot-and-mouth disease virus antigen;

OP _CA - average optical density value of antigen control;

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

The stated objective is achieved by the fact that the proposed test system uses two calculated and three standard internal controls: antigen control (AC); conjugate control (CC); positive and weakly positive blood serum containing specific antibodies to the antigen of the SAT-2/XIV/2023 strain of foot-and-mouth disease virus; negative blood serum that does not have specific antibodies, which allows for a more accurate interpretation of the reaction results.

The proposed test system is intended mainly for screening mass studies using the point method of sample dilution, but it is possible to study sera using the method of successive dilutions with determination of the end point of titration or serum titer. This enzyme immunoassay test system for assessing the antigenic and immunogenic activity of the foot-and-mouth disease vaccine based on the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus (the proposed invention) is also intended for assessing the antigenic and immunogenic activity of foot-and-mouth disease vaccines; monitoring examination of vaccinated livestock for the intensity of immunity to foot-and-mouth disease; post- and pre-sale certification of farm animals for the purposes of international and domestic trade; examination of livestock not vaccinated against foot-and-mouth disease in order to confirm or obtain the status of a foot-and-mouth disease-free zone for regions without vaccination; retrospective diagnostics of foot-and-mouth disease, including, in combination with studies for the presence of antibodies to non-structural proteins of the foot-and-mouth disease virus, identifying animals that have recovered from the disease or animals that are carriers of the virus.

The enzyme immunoassay test system for assessing the antigenic and immunogenic activity of the foot-and-mouth disease vaccine based on the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus is a specific and non-specific component for carrying out the indirect liquid-phase blocking ELISA reaction.

Immunospecific components:

K1 – Sensitizing antibodies, rabbit IgG against the intact antigen of the SAT-2/XIV/2023 strain of foot-and-mouth disease virus, lyophilized, volume 0.5 ^cm3 – 1 bottle;

K2 - Antigen of the SAT-2/XIV/2023 strain of foot-and-mouth disease virus, highly purified and concentrated, lyophilized, volume 0.5 ^cm3 - 1 bottle;

K3 - Detector antibodies, guinea pig IgG against the intact antigen of the SAT-2/XIV/2023 strain of foot-and-mouth disease virus, lyophilized, volume 0.5 ^cm3 - 1 bottle;

K4 - Control 1, positive blood serum against the antigen of the SAT-2/XIV/2023 strain of foot-and-mouth disease virus, lyophilized, volume 0.2 ^cm3 - 1 bottle;

K5 - Control 2, positive blood serum against the antigen of the SAT-2/XIV/2023 strain of foot-and-mouth disease virus, lyophilized, volume 0.2 ^cm3 - 1 bottle;

K6 - Control 3, normal blood serum not containing antibodies to foot-and-mouth disease virus, lyophilized, volume 0.2 ^cm3 – 1 bottle;

K7 – Conjugate, immunoperoxidase conjugate of secondary antibodies against guinea pig IgG, lyophilized or native, volume 0.1 ^cm3 – 1 vial;

K8 – Blocking serum, non-immune, lyophilized, volume 4.0 ^cm3 – 1 bottle.

Non-specific components:

K9 – Salts for carbonate-bicarbonate buffer solution, powder in capsule or tablet, weight 0.215-0.43 g – 1 bottle;

K10 – Buffer solution 20x, liquid, volume 100.0 ^cm3 - 1 bottle;

K11 – Chromogenic substrate ABTS, liquid, volume 25.0 ^cm3 - 1 bottle;

K12 - Stop solution, 1% sodium dodecyl sulfate, liquid, volume 25.0 ^cm3 - 1 bottle.

The test system also includes:

- polystyrene 96-well plate for enzyme immunoassay, whole or stripped – 3 pcs.;

- polymer tablet – 3 pcs.;

- instructions for use – 1 copy.

The technical result of the invention consists in developing a sensitive and specific enzyme immunoassay test system based on the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus, designed to assess the antigenic and immunogenic activity of the foot-and-mouth disease vaccine. In addition, the lyophilized immunospecific components are stable and retain their activity for at least 3 years (observation period), which is an advantage in using this test system.

The essence of the invention is reflected in the graphic images:

Fig. 1 – Epizootic situation in the world for foot-and-mouth disease (type SAT 2).

Fig. 2 – Amino acid sequences of the polypeptide VP ₁ of foot-and-mouth disease virus serotype SAT 2 topotypes XIV and VII from the NCBI or FGBU “ARRIAH” database (up to 216 aa). Note: the GH-loop zone is highlighted in red .

Fig. 3 – Location of the “SAT-2/XIV/2023” strain of foot-and-mouth disease virus on the phylogenetic tree of foot-and-mouth disease virus serotype SAT 2. The dendrogram is based on a comparison of the nucleotide sequences of the VP ₁ gene. Note: topotypes I-XIV are highlighted in square brackets.

Fig. 4 - Isolation of the 146S antigen of the foot-and-mouth disease virus strain "SAT-2/XIV/2023" during fractionation of the antigen precipitate during ultracentrifugation in a sucrose density gradient: A - sedimentation profile of the antigen of the foot-and-mouth disease virus strain "SAT-2/XIV/2023" at a wavelength of 260 nm; B - electropherogram of the sucrose density gradient fractions including the structural proteins VP ₁ , VP ₂ , VP ₃ of the foot-and-mouth disease virus strain "SAT-2/XIV/2023".

Fig. 5 - Electrophoresis in 12% polyacrylamide gel of foot-and-mouth disease virus antigen of strain "SAT-2/XIV/2023": antigen precipitate before fractionation by ultracentrifugation in a sucrose density gradient; 146S antigen.

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

SEQ ID NO:1 represents the nucleotide sequence of the gene encoding the VP ₁ protein of the strain "SAT-2/XIV/2023" of the genotype SAT2/XIV foot-and-mouth disease virus;

SEQ ID NO:2 represents the amino acid sequence of protein VP ₁ of the strain "SAT-2/XIV/2023" of the genotype SAT-2/XIV foot-and-mouth disease virus.

The main material for the study is individual animal blood serum samples without signs of hemolysis and bacterial contamination, manifested by turbidity of the sample or sediment formation. Animal blood serum may be stored before ELISA testing at a temperature of (5±3)°C for 24-48 hours after collection; for longer storage, samples are frozen at a temperature of minus (18±2)°C. Blood serum samples are thawed before testing. Multiple freezing and thawing of samples is not recommended.

Before starting work, lyophilized components K1-K8 are restored to the volume indicated on the label, for which the required amount of distilled water is added to the contents of the vials. The conjugate in its native form is ready for use.

Prepare working solutions:

- Solution No. 1. Carbonate-bicarbonate buffer solution for sensitization of tablets. In accordance with the information indicated on the label, the contents of the capsule or tablet (K9) are dissolved in 100 (50) cm ³ of distilled water and mixed thoroughly.

- Solution No. 2. The solution is used for interstage washing of plates and preparation of samples for research, and also as a basis for preparing a working buffer solution for diluting detector antibodies and conjugate. For this, the contents of the vial with the concentrate (20x) of the buffer solution (K10) are poured into a measuring vessel, distilled water is added to 2000 cm ³ and mixed, or the required amount of solution No. 2 is made in the appropriate proportions.

- Solution No. 3. The working buffer solution for diluting the detector antibodies and conjugate is prepared from solution No. 2 with the addition of 10% blocking serum. For this purpose, 4.0 cm ³ of blocking serum, reconstituted from lyophilized preparation K8 by adding 4.0 cm ³ of distilled water to the contents of the vial with the serum, should be added to 36 cm ³ of solution No. 2. Prepared immediately before use.

Chromogenic substrate ABTS , K11, ready to use.

Stop solution . The solution that stops the coloring reaction (K12) is ready for use. At a temperature of (5±3)°C, precipitation is possible, which dissolves when heated to a temperature of (22±3)°C and then shakes the contents of the bottle, which does not affect the quality of the reagent.

The reaction of the indirect liquid-phase blocking variant of the enzyme-linked immunosorbent assay for assessing the antigenic and immunogenic activity of the foot-and-mouth disease vaccine based on the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus is performed in stages in the following sequence:

- Sensitization of plates. The solution of sensitizing antibodies is prepared in the dilution indicated on bottle K1, in solution No. 1 (carbonate-bicarbonate buffer solution). 0.1 ^cm3 (100 μl) of the sensitizing antibody solution is added to all wells of a polystyrene 96-well plate for enzyme immunoassay, covered with a lid and incubated for (18±2) h at a temperature of (5±3)°C.

- Washing of the wells of the plate. Washing is carried out using automatic devices or manually. In this case, the contents of the wells of the plate are removed, then the wells are filled with solution No. 2 and dumped by vigorous shaking. The procedure is repeated twice.

- Liquid phase reaction. The antigen is reacted with the test and control blood serum samples. The test and control samples are arranged on the plate according to the recommended scheme. Positive blood serum (K4) is used as control 1, positive blood serum (K5) as control 2, and normal blood serum (K6) as control 3. First, 1:16 dilutions of the samples are prepared in solution No. 2. For this purpose, 0.047 cm3 (47 μl) of buffer solution and 0.003 ^cm3 (3 μl) of sample are added to all wells of the round-bottomed polymer plate, except for wells A1, B1, C1, D1, which are left for the conjugate control (CC) and the antigen control (AC). 0.05 ^cm3 (50 μl) ^of buffer solution are added to wells A1, B1, C1, D1. Then, 0.05 ^cm3 (50 µl) of the working dilution of antigen in solution No. 2 is added to all wells of the plate in accordance with the information indicated on the label (final sample dilution 1:32). The plate is covered with a lid and incubated for (18±2) h at a temperature of (5±3)°C.

- Antigen capture. 0.05 ^cm3 (50 µl) of the mixture of control and test samples and antigen are added to the washed wells of the sensitized plate, the plate is covered with a lid and incubated for (60±5) min at a temperature of (37±2)°C.

- Washing the wells of the plate. The procedure is repeated 3 times, as described above.

- Addition of detector antibodies. In all wells of the plate, except for wells A1 and B1 with KK, where 0.05 ^cm3 (50 μl) of solution No. 3 is added, add 0.05 ^cm3 (50 μl) of the working dilution of detector antibodies in solution No. 3 in accordance with the dilution indicated on the label of the K3 bottle, cover the plate with a lid and incubate for (60±5) min at a temperature of (37±2)°C.

- Washing the wells of the plate. The procedure is repeated 3 times, as described above.

- Addition of the conjugate. Add 0.05 ^cm3 (50 μl) of the working dilution of the conjugate in solution No. 3 to all wells of the plate in accordance with the dilution indicated on the label of the K7 vial, close the plate with a lid and incubate for (60±5) min at a temperature of (37±2)°C.

- Washing the wells of the plate. The procedure is repeated 3 times, as described above.

- Staining. Add 0.05 ^cm3 (50 µl) of chromogenic ABTS substrate to each well of the plate, cover with a lid and keep for (15±5) min at a temperature of (22±3)°C.

- Stopping the reaction. The reaction is stopped by adding 0.05 ^cm3 (50 µl) of stop solution to each well of the plate.

The results of the analysis are taken into account instrumentally. Immediately after stopping the reaction, the optical density (OD) of the reaction products in each well is measured at a wavelength of 405-415 nm using a spectrophotometer (reader) for microplates with a vertical light beam. The OD values of the control and test samples are converted into the value of the percentage of inhibition (PI) using 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 foot-and-mouth disease virus antigen;

OP _CA - average optical density value of antigen control;

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

The reaction is considered reliable if it meets the following criteria:

- antigen control has an OP value of 0.5 optical units or higher;

- the difference between the values of OP _KA and OP _KK is not less than 0.4 optical units;

- control 1 has a PI value>75%;

- control 2 has a PI value>50%;

- control 3 has a PI value of <50%.

Samples demonstrating a PI value of ≥50% are considered positive, and the animals from which these serum samples were obtained are considered immune to foot-and-mouth disease genotype SAT2/XIV. A PI value of <50% is negative, indicating the absence of specific antibodies to foot-and-mouth disease virus genotype SAT2/XIV in the blood serum of the animals examined.

As a result of the studies, optimal working dilutions of specific reagents were determined. The activity of sensitizing antibodies in the ELISA reaction was 1:1000; antigen of the SAT-2/XIV/2023 strain of foot-and-mouth disease virus of the SAT2/XIV genotype - 1:1000, or 0.55 μg/cm ³ ; detector antibodies - 1:5000, commercial anti-species conjugate - 1:1500, or 0.75 μg/cm ³ ; control samples of blood serum demonstrating PI values in the range specified above were selected. Upon receipt of new batches of components, one or more, it is necessary to determine their working concentration for the ELISA reaction, providing standard values of the control samples, and validate the test system.

The developed test system makes it possible to test 86 blood serum samples simultaneously in the format of one 96-well plate without taking into account controls when setting up a reaction in one repetition or 43 studies in two repetitions (recommended).

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

Example 1. Selection of a strain of foot-and-mouth disease virus serotype SAT 2 for the development of a test system that allows for the assessment of the antigenic and immunogenic activity of an anti-foot-and-mouth disease vaccine that includes the antigen of the production strain “SAT-2/XIV/2023” of the foot-and-mouth disease virus.

When creating diagnostic test systems for foot-and-mouth disease, developers strive for serotype specificity of ELISA. Indeed, within a serotype, the foot-and-mouth disease virus of different genetic lines, as a rule, exhibits serological kinship. However, when examining primarily vaccinated young animals, test systems for detecting antibodies to the foot-and-mouth disease virus of a certain serotype from different manufacturers may demonstrate strain/genotypic specificity. This is of great importance when assessing the antigenic and immunogenic activity of the foot-and-mouth disease vaccine. The tool should be carefully selected to obtain the most objective and reliable information.

Before developing a test system for assessing the antigenic and immunogenic activity of foot-and-mouth disease vaccines of serotype SAT 2 topotype XIV, the Federal State Budgetary Institution "ARRIAH" created two test systems for assessing humoral immunity to foot-and-mouth disease of serotype SAT 2 topotype VII based on the foot-and-mouth disease virus strains SAT2/LIB/2012 and SAT2/ERI/98, which showed a high degree of kinship and, as a result, interchangeability. It was assumed that these test systems would be able to provide full control in ELISA of the effectiveness of a vaccine including the antigen of the "SAT-2/XIV/2023" strain of foot-and-mouth disease virus. The data on genetic and serological correspondence of strains and isolates of foot-and-mouth disease virus of VII and XIV topotypes of serotype SAT 2 were analyzed, and the feasibility of developing this test system for foot-and-mouth disease of serotype SAT 2 topotype XIV was considered.

To identify the genetic relationship (homology) between topotypes VII and XIV, a comparison of the amino acid sequences of the VP protein was performed.₁foot-and-mouth disease virus serotype SAT 2. The alignment was performed according to information obtained from NCBI (https://www.ncbi.nlm.nih.gov/) [18] and the FGBU "ARRIAH" database: SAT2/ETH/2/2022 (GenBank: WKE35517.1); SAT2/JOR/11/2023 (GenBank: WUR05443.1); SAT-2/XIV/2023(database of the Federal State Budgetary Institution "All-Russian Research Institute of Animal Health"); SAT2/ETH/2/91 (GenBank: WKE35516.1); SAT2/ERI/4/98 (GenBank: AAR09103); SAT2/LIB/39/2012 (GenBank: AFU55197.1); SAT2/EGY/Ismalia/2018 (GenBank: QZE50286.1); SAT2/EGY/Beni-Suef-2/2018 (GenBank: QEI49588.1); SAT2/EGY/Dakahlia/2017 (GenBank: AXR97922.1) (Fig. 2).

As shown in Fig. 2, the vaccine strain "SAT-2/XIV/2023" of foot and mouth disease virus has 100% homology with the foot and mouth disease virus isolate isolated in the Hashemite Kingdom of Jordan in 2023 (SAT2/JOR/11/2023) in the amino acid sequence of the VP ₁ polypeptide, both viruses differ from the isolate SAT2/ETH/2/2022 isolated in Ethiopia in 2022 by one amino acid substitution in the GH-loop region and 20 substitutions from the Ethiopian isolate SAT2/ETH/2/91. These viruses belong to topotype XIV of serotype SAT 2 and differ significantly from isolates of foot-and-mouth disease virus of serotype SAT 2 of topotype VII, which directly affects the antigen correspondence of strains of foot-and-mouth disease virus of serotype SAT 2 belonging to different topotypes. The phylogenetic tree of foot-and-mouth disease virus of serotype SAT 2 is presented in Fig. 3, based on comparison of nucleotide sequences of the VP ₁ gene, clearly demonstrates topotypic (genotypic) differences between strains of the virus.

When studying the antigenic correspondence of the vaccine strain "SAT-2/XIV/2023" (topotype XIV) of the foot-and-mouth disease virus with the foot-and-mouth disease virus strains SAT2/LIB/2012 (topotype VII) and SAT2/ERI/98 (topotype VII) in the neutralization reaction on the IB-RS-2 cell culture, a low relationship was found between the strains of different topotypes from 0.06 to 0.16. At the same time, the strains SAT2/LIB/2012 and SAT2/ERI/98 demonstrated close relationship with each other, 0.36 and 0.69, respectively (Table 1), which indicated good cross-protection between these foot-and-mouth disease viruses.

Thus, when developing an enzyme immunoassay test system to assess the antigenicity and immunogenicity of the vaccine against foot-and-mouth disease of the SAT2/XIV genotype, the production strain “SAT-2/XIV/2023” of the foot-and-mouth disease virus was selected.

Example 2. Obtaining immunospecific components of an enzyme immunoassay test system for assessing the antigenic and immunogenic activity of an anti-foot-and-mouth disease vaccine based on the SAT-2/XIV/2023 strain of foot-and-mouth disease virus.

In obtaining specific reagents for the liquid-phase blocking ELISA variant for detecting antibodies to structural proteins of the foot-and-mouth disease virus of the SAT2/XIV genotype, special attention was paid to the isolation of the 146S component from the inactivated virus-containing suspension of the BHK-21 cell culture (146S antigen of the foot-and-mouth disease virus of the SAT2/XIV genotype). This is an intact antigen with a sedimentation coefficient of 146S, i.e. an antigen with an unchanged structure, representing virions that have lost their infectivity during inactivation. This antigen is a capsid with inactivated RNA enclosed in it. The integrity of the capsid ensures the immunogenicity of the foot-and-mouth disease vaccine, since the surface polypeptides VP ₁ -VP ₃ produce virus-neutralizing strain/serotype-specific antibodies. The use of highly purified 146S antigen in the ELISA reaction during the "liquid phase" during which an immune complex is formed with specific antibodies in the test sample, as well as for the immunization of rabbits and guinea pigs when obtaining catching (sensitizing) and detector antibodies, respectively, allows determining the level of humoral immunity against foot-and-mouth disease with the highest possible reliability. The presence of inert ballast proteins in the form of cellular debris and serum albumin in a partially purified antigen or antigen-containing cell suspension may lead to distortion of the reaction results.

The antigen of the SAT-2/XIV/2023 strain of foot-and-mouth disease virus for obtaining immunospecific components of the ELISA reaction: antigen, capturing (sensitizing) and detector antibodies, was isolated from an inactivated virus-containing suspension of the BHK-21 cell culture according to the following scheme.

In the first stage, the antigen raw material was freed from cellular debris by low-speed centrifugation (Avanti J-26 XP, Beckman Coulter, USA) for 30 minutes. The supernatant was decanted and used to precipitate the viral antigen in the presence of 8% polyethylene glycol with a molecular weight of 6000 (PEG6000) and 0.85% NaCl for 18-20 hours at a temperature of (6±2)°C. The precipitated antigen was sedimented in an Avanti J-26 XP centrifuge, Beckman Coulter, USA, at 6000 rpm, a temperature of 4°C for 60 minutes. The supernatant was removed, the sediment was resuspended in phosphate-buffered saline (PBS), pH 7.4, 1/500 of the original volume of the original raw material. The resulting suspension was then thoroughly homogenized with 50% chloroform and fractionated at 3000 rpm, 4°C for 15 minutes in an Allegra X-22R Centrifuge, Beckman Coulter, USA. The upper aqueous fraction was an intermediate substance in the form of a concentrated, partially purified antigen, designated hereinafter as the antigen precipitate of the SAT-2/XIV/2023 strain of foot-and-mouth disease virus (SAT2/XIV _antigen precipitate). Then, SAT2/XIV _PC Ag were fractionated in a step sucrose density gradient consisting of successive layers of 20%, 30%, 40%, 50% sucrose in PBS, in an Optima L-80 XP Ultracentrifuge, Beckman Coulter, USA, at 24,000 rpm, 4 ⁰ C for 3 hours. The bulk of the 146S particles accumulated at the interface between the 30% and 40% sucrose layers as an opalescent band. Gradient fractions (1 ml each) were analyzed spectrophotometrically at a wavelength of 260 nm to construct the sedimentation profile of SAT2/XIV _PC Ag (Fig. 4A) and during electrophoretic separation of protein molecules in a 12% polyacrylamide gel (Fig. 4B). For the 146S antigen, fractions with the highest accumulation of structural polypeptides VP ₁ -VP ₃ and the lowest content/absence of impurities were selected. As a result, after combining the fractions, 146S antigen SAT2/XIV with a protein concentration of approximately 0.55 mg/ml was obtained. Fig. 5 shows an electropherogram of the antigen of the SAT-2/XIV/2023 strain of foot-and-mouth disease virus in a 12% polyacrylamide gel: antigen precipitate before fractionation by ultracentrifugation in a sucrose density gradient; 146S antigen (146S-Ag SAT2/XIV) of foot-and-mouth disease virus.

The obtained 146S-Ag SAT2/XIV was used to manufacture immunospecific components of the liquid-phase blocking ELISA reaction: capture and detector antibodies, foot-and-mouth disease virus antigen.

Rabbits and guinea pigs were immunized twice with 146S-Ag SAT2/XIV at a dose of approximately 0.3 and 0.15 mg per head, respectively. The antigen for administration was mixed with Montanide ISA-206 adjuvant in equal proportions. Bleeding was performed on the 35th day after vaccination.

The test system for assessing the antigenic and immunogenic activity of foot-and-mouth disease vaccines based on the SAT-2/XIV/2023 strain of foot-and-mouth disease virus contains immunospecific reagents in the following working dilutions: capture antibodies - 1:1000, detector antibodies - 1:5000, foot-and-mouth disease virus antigen - 1:1000 or 0.55 μg/ml.

Example 3. Calculation of the positive-negative threshold for the qualitative assessment of the results of a study of animal blood serum samples using an enzyme immunoassay test system to assess the antigenic and immunogenic activity of the foot-and-mouth disease vaccine based on the strain "SAT-2/XIV/2023" foot-and-mouth disease virus.

When interpreting the results of the ELISA reaction, it is necessary to establish criteria for qualitative assessment, for which a cut-off point, a positive-negative threshold (PNT), is determined, which allows qualifying the studied biomaterial samples as positive or negative with respect to a specific infectious agent. In competitive or blocking enzyme immunoassay test systems for detecting specific antibodies to the foot-and-mouth disease virus from different manufacturers, the reference point is usually the percentage of inhibition (PI) or S/N ratio of 50%. However, manufacturers sometimes use other values for PNT in accordance with the sensitivity and specificity of their test systems. For example, in the SAT 2 FMD kit manufactured by IZSLER, which is compared with the proposed SAT2/XIV FGBU "ARRIAH" FMD test system, the threshold is set at 70%.

In the proposed test system for assessing the antigenic and immunogenic activity of foot-and-mouth disease vaccines based on the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus, the qualitative analysis of the ELISA reaction results is based on the PNP of 50% PI. The calculation was based on the data obtained during testing of 176 blood serum samples from clinically healthy cattle and pigs not vaccinated against foot-and-mouth disease. The mean PI value and standard deviation from the mean (SD) were obtained. The mean PI value for this sample of blood serum samples (n=176) was 21.09%, SD – 14.67%. The positive-negative threshold was calculated using the formula PNP=PI _mean +2SD, which was 50.43%.

Thus, the value of the positive-negative threshold for the qualitative characteristics of blood serum samples studied in the proposed test system for assessing the antigenic and immunogenic activity of foot-and-mouth disease vaccines based on the SAT-2/XIV/2023 strain of foot-and-mouth disease virus corresponded to the predicted 50% PI, which indicated the optimal choice of working dilutions of immunospecific components that determine the accuracy and objectivity of the analysis.

Example 4. Use of an enzyme immunoassay test system to assess the antigenic and immunogenic activity of an anti-foot-and-mouth disease vaccine containing the antigen of the foot-and-mouth disease virus strain "SAT-2/XIV/2023" foot-and-mouth disease virus.

The proposed test system was tested in an experiment on cattle to check the antigenicity and immunogenicity of the monovalent adsorbed vaccine against foot-and-mouth disease SAT2/XIV in comparison with other test systems for foot-and-mouth disease of serotype SAT 2 (Table 2). Fifteen bulls were immunized once with the vaccine in whole form, in dilutions of 1:4 and 1:16, 5 individuals for each vaccine dilution. One control animal was not vaccinated. Blood for ELISA and RN testing was collected from all animals on days 0, 3, 7, 14, and 21 after administration of the vaccine against foot-and-mouth disease of genotype SAT2/XIV. Blood serum samples were tested for the presence of antibodies to foot-and-mouth disease virus serotype SAT 2 in the enzyme immunoassay test systems: SAT2/XIV-ARRIAH based on the SAT-2/XIV/2023 foot-and-mouth disease virus strain (proposed invention), SAT2/VII-ARRIAH based on the SAT2/LIB/39/2012 foot-and-mouth disease virus strain, SAT2-IZSLER, and in the SAT-2/XIV/2023 foot-and-mouth disease virus neutralization reaction. As can be seen from Table 2, the efficiency of the SAT2/XIV-ARRIAH test system based on the SAT-2/XIV/2023 foot-and-mouth disease virus strain (proposed invention) in recording the production of specific antibodies was higher than that of the other test systems under consideration. As early as on the 7th day, one seropositive individual was detected in three groups of vaccinated cattle, in other reactions, specific antibodies were detected only on the 14th day. The trend persisted on the 21st day after vaccination. As a result, the number of positively reacting animals established in ELISA and RN on the 14-21st days after the administration of different doses of the vaccine was: SAT2/XIV-ARRIAH test system based on the SAT-2/XIV/2023 strain of foot-and-mouth disease virus (the proposed invention) - 22/30 (73.3%), SAT2/VII-ARRIAH - 13/30 (43.4%), SAT2-IZSLER - 8/30 (26.7%), SAT2/XIV-RN - 11/30 (36.7%), which indicated an undeniable advantage in diagnostic sensitivity of the proposed test system.

To confirm the objectivity of this enzyme immunoassay test system in assessing the antigenic and immunogenic activity of the foot-and-mouth disease vaccine based on the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus (proposed invention), 163 blood serum samples from pigs and cattle were analyzed. The analysis was carried out using the following test systems: based on the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus (proposed invention), SAT2/VII-ARRIAH, SAT2-IZSLER. The results of parallel studies of blood sera in ELISA before and after vaccination with a whole dose against foot-and-mouth disease of the genotypes SAT2/XIV (vaccine based on the strain "SAT-2/XIV/2023" of the foot-and-mouth disease virus) and SAT2/VII (vaccines based on the strains SAT2/LIB/39/2012 and SAT2/ERI/98), as well as after infection with the foot-and-mouth disease virus strain SAT2/ERI/98 are presented in Table 3. As the analysis of Table 3 shows, the test systems demonstrated pronounced genotype specificity when testing this sample set. Cattle blood serum against the antigen of the SAT-2/XIV/2023 strain of foot-and-mouth disease virus when using a test system for assessing the antigenic and immunogenic activity of an anti-foot-and-mouth disease vaccine, including the antigen of the SAT-2/XIV/2023 strain of foot-and-mouth disease virus (the proposed invention), had a positive status in 100% of cases, in the SAT2/VII-ARRIAH and SAT2-IZSLER ELISA reactions - in 80% and 60%, respectively. While the seropositivity of animals with respect to foot-and-mouth disease serotype SAT 2 topotype VII was established in the SAT2/VII-ARRIAH and SAT2-IZSLER test systems at 88% and 91%, respectively, in the test system for assessing the antigenic and immunogenic activity of the foot-and-mouth disease vaccine, including the antigen of the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus (the proposed invention) – at 81%.

Thus, the proposed enzyme immunoassay test system for assessing the antigenic and immunogenic activity of the foot-and-mouth disease vaccine, including the antigen of the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus, can be used to assess the effectiveness of the vaccine and the intensity of immunity against foot-and-mouth disease of the SAT2/XIV genotype.

Example 5. Determination of the diagnostic characteristics of an enzyme immunoassay test system for assessing the antigenic and immunogenic activity of an anti-foot-and-mouth disease vaccine containing the antigen of the SAT-2/XIV/2023 strain of foot-and-mouth disease virus.

During the validation of the proposed test system, the main diagnostic characteristics such as sensitivity, specificity, accuracy and kappa criterion were determined, as described in the methodology [19]. Table 4 shows the data of statistical processing of the ELISA testing results for 113 samples of bovine blood serum obtained against the foot-and-mouth disease virus antigen of the SAT2/XIV genotype. As can be seen from Table 4, the high values of diagnostic sensitivity of 90.0%, diagnostic specificity of 98.1%, diagnostic accuracy of 93.8% of the enzyme immunoassay test system for assessing the antigenic and immunogenic activity of the foot-and-mouth disease vaccine containing the antigen of the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus (the proposed invention) determined a high degree of consistency of the ELISA reaction results with the known diagnostic status of the animals examined (k-criterion - 0.876).

Sources of information used in drafting the application for the issuance of a patent of the Russian Federation for the invention "Enzyme-linked immunosorbent assay system for assessing the antigenic and immunogenic activity of an anti-foot-and-mouth disease vaccine based on the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus":

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Table 1

Antigenic relationship between strains of foot and mouth disease virus serotype SAT 2 in the neutralization reaction, r ₁

Samples
blood serum r _1avg. in the reaction of neutralization of foot-and-mouth disease virus SAT2/LIB/2012 SAT2/ERI/98 «SAT-2/XIV/2023» SAT2/LIB/2012 1.0 0.36 0.06 SAT2/ERI/98 0.69 1.0 0.16 «SAT-2/XIV/2023» 0.14 0.16 1.0

Note: if r ₁ ≥0.3 - close relationship;

r ₁ <0.3 – low kinship

Table 2

Results of the study in ELISA and RN of blood serum samples of cattle vaccinated against foot-and-mouth disease strain "SAT-2/XIV/2023"

(sorbed monovalent vaccine)

Blood serum samples Dilution of the vaccine Test systems used SAT2/XIV-VNIIZZH
(proposed invention )
PI _pl ≥50% SAT2/VII- VNIIZh PI _pl ≥50% SAT2-IZSLER
PI _pl ≥70% RN
VY strain
«SAT-2/XIV/2023»
T _pl VNA≥5.5log ₂ PI _average pl/n PI _average pl/n PI _average pl/n T _avg. pl/n Skrs 0 dpv before vaccination 23.2 0/16 33.0 0/16 22.3 0/16 0 0/16 Skrs 3 dpv whole 16.1 0/5 22.2 0/5 2.4 0/5 0 0/5 1:4 9.8 0/5 29.7 0/5 3.2 0/5 0 0/5 1:16 11.1 0/5 36.1 0/5 2.9 0/5 0 0/5 non-vacc.control 19.4 0/1 18.0 0/1 2.8 0/1 0 0/1 Skrs 7 dpv whole 35.7 1/5 32.7 0/5 12.2 0/5 3.5 0/5 1:4 26.8 1/5 36.9 0/5 16.9 0/5 2.8 0/5 1:16 15.3 1/5 23.7 1/5 25.7 0/5 1.0 0/5 non-vacc.control 13.8 0/1 17.7 0/1 19.2 0/1 0 0/1 Skrs 14 dpv whole 75.0 5/5 65.9 4/5 72.4 3/5 5.3 3/5 1:4 62.1 4/5 44.4 2/5 48.1 1/5 4.6 2/5 1:16 40.7 2/5 36.6 1/5 32.0 0/5 3.4 1/5 non-vacc.control 22.3 0/1 22.1 0/1 11.3 0/1 ≤1.5 0/1 Skrs 21 dpv whole 74.5 5/5 63.9 4/5 71.7 3/5 5.4 3/5 1:4 63.4 4/5 32.0 1/5 44.2 1/5 4.4 1/5 1:16 42.6 2/5 24.7 1/5 37.5 0/5 3.2 1/5 non-vacc.control 15.7 0/1 9.5 0/1 7.3 0/1 ≤1.5 0/1

Note: PI – percentage of inhibition;

T _pl VNA – minimum positive titer of virus-neutralizing antibodies;

pl/n – the number of positively reacting individuals to the total number of animals in the group;

dpv – days after vaccination

RN – neutralization reaction

Table 3

Results of the ELISA study of blood serum samples from cattle and pigs before and after vaccination/after infection against foot and mouth disease serotype SAT 2

Samples
blood serum Test systems used SAT2/XIV-VNIIZZH
(proposed invention ) PI _pl ≥50% SAT2/VII-VNIIZZH
PI _pl ≥50% SAT2-IZSLER
PI _pl ≥70% PI _average pl/n SP,% PI _average pl/n SP,% PI _average pl/n SP,% Ssvin. 0 dpv 23.2 0/16 - 13.9 0/16 - 22.3 0/16 - Skrs 0 dpv 25.1 0/37 - 30.3 0/37 - 20.2 0/37 - Sswine 16-28 dpv SAT2/LIB/2012 68.2 28/30 93.3 83.7 29/30 96.7 90.2 29/30 96.7 Skrs 14-35 dpv SAT2/LIB/2012 70.1 14/15 93.3 89.5 15/15 100,0 87.6 15/15 100,0 Skrs 21-45 dpv/dpi SAT2/ERI/98 59.5 23/35 65.7 80.3 27/35 77.1 89.1 29/35 82.9 Skrs 14-21 dpv “SAT-2/XIV/2023” 76.8 30/30 100,0 63.8 24/30 80.0 72.6 18/30 60.0

Note: PI – percentage of inhibition;

pl/n – the number of positively reacting individuals to the total number of animals in the group;

SP – seropositivity (number of animals reacting positively);

dpv/dpi – days after vaccination/days after infection

Table 4

Diagnostic characteristics of test systems for detection of antibodies to structural proteins of foot-and-mouth disease virus serotype SAT 2, determined for genotype SAT2/XIV

n=113

Used
test systems Diagnostic characteristics () Sensitivity Specificity Accuracy k-criterion* SAT2/XIV-VNIIZZH
(proposed invention ) 90.0%
(79.5%-96.2%) 98.1%
(89.9%-100.0%) 93.8%
(87.7%-97.5%) 0.876 SAT2/VII-VNIIZZH 55.0%
(41.6%-67.9%) 98.1%
(89.9%-100.0%) 75.2%
(66.2%-82.9%) 0.516 SAT2-IZSLER 40.0%
(27.6%-53.5%) 100.0%
(93.3%-100.0%) 68.1%
(58.7%-76.6%) 0.385 RN VYA strain
«SAT-2/XIV/2023» 45.0%
(29.3% - 61.5%) 100.0%
(93.3%-100.0%) 76.3%
(66.4%-84.5%) 0.483

Note: *k-criterion – agreement of the results of testing individual blood serum samples in ELISA and RN with the diagnostic status of animals:

<0 – no agreement;

0-0.20 – insignificant;

0.21-0.40 – weak;

0.41-0.60 – moderate;

0.61-0.80 – significant;

0.81-1.00 – high.

Claims (5)

1. An enzyme immunoassay test system for assessing the antigenic and immunogenic activity of the foot-and-mouth disease vaccine based on the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus, containing: immunospecific reagents: highly purified intact antigen of the strain "SAT-2/XIV/2023" genotype SAT2/XIV foot-and-mouth disease virus, lyophilized with ELISA activity of 1:1000; sensitizing antibodies, Rabbit IgG against the strain "SAT-2/XIV/2023" genotype SAT2/XIV of foot-and-mouth disease virus, lyophilized with ELISA activity of 1:1000; detector antibodies, guinea pig IgG against the strain "SAT-2/XIV/2023" genotype SAT2/XIV of foot-and-mouth disease virus, lyophilized with ELISA activity of 1:5000; control 1 - positive blood serum to the antigen of the strain "SAT-2/XIV/2023" genotype SAT2/XIV of foot-and-mouth disease virus with an inhibition rate of more than 75%, lyophilized; control 2 - positive blood serum to the antigen of the strain "SAT-2/XIV/2023" genotype SAT2/XIV of foot-and-mouth disease virus with an inhibition rate of more than 50%, lyophilized; control 3 - normal blood serum that does not contain antibodies to foot-and-mouth disease virus, with an inhibition rate of less than 50%, lyophilized; conjugate - immunoperoxidase conjugate of secondary antibodies against guinea pig IgG, lyophilized; non-immune blocking serum, lyophilized; non-specific components: CBB - salts for carbonate-bicarbonate buffer solution, pH 9.5-9; buffer solution - 20x, pH 7.4-7.6;chromogenic substrate ABTS; stop solution - 1% sodium dodecyl sulfate solution, characterized in that, when obtaining immunospecific components, an inactivated antigen of the foot-and-mouth disease virus strain "SAT-2/XIV/2023" of the foot-and-mouth disease virus is used and the level of post-vaccination immunity is determined by the amount of specific antibodies in the blood serum of immunized cattle, expressed through the percentage of inhibition (PI) in accordance with the formula: PI = (1 - (OD _sample - OD _CC ) / (OD _CA - _{OD CC} )) x 100%, while the titer of anti-foot-and-mouth disease antibodies is determined as the maximum dilution of blood serum for which the percentage of inhibition is ≥50%. 2. The test system according to item 1, characterized in that it is specific and sensitive, allowing for a rapid, within 4 hours, assessment of the antigenic and immunogenic activity of the foot-and-mouth disease vaccine based on the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus.