RU2818811C1 - Test system for determining titre of antibodies against structural proteins of foot-and-mouth disease virus of "a/tanzania/2013" strain using liquid-phase enzyme immunoassay - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a test system for determining the titre of antibodies against structural proteins of the A/Tanzania/2013 strain of the foot-and-mouth disease virus using a liquid-phase enzyme immunoassay.

EFFECT: technical result consists in the development of a modern, specific against structural proteins of the A/Tanzania/2013 strain of the A/AFRICA/G-I genotype and a sensitive test system, intended for determination of antibody titre against structural proteins of specified strain of foot-and-mouth disease virus by method of liquid-phase blocking indirect "sandwich"-variant of ELISA on the basis of application of inactivated virus of foot-and-mouth disease of A/Tanzania/2013 strain of genotype A/AFRICA/G-I and during production of immunospecific reaction components.

2 cl, 3 dwg, 6 tbl, 6 ex

Description

The invention relates to the field of veterinary virology and biotechnology, in particular, to the creation of a test system for determining the titer of antibodies against the structural proteins of the foot-and-mouth disease virus strain “A/Tanzania/2013” using a liquid-phase enzyme-linked immunosorbent assay.

Foot-and-mouth disease virus is a member of the genus Aphthovirus of the family Picornaviridae and includes the following seven highly different serotypes: O, A, Asia-1, C, SAT-1, SAT-2, SAT-3 [1]. At the same time, serotype A is antigenically very diverse. Complete virus particles with a sedimentation coefficient of 146S consist of a single-stranded positive-sense RNA genome and 60 copies of each of the following four structural proteins: VP ₁ (1D gene), VP ₂ (1B gene), VP ₃ (1C gene) and VP ₄ (1A gene) [2].

For the foot-and-mouth disease pathogen, which is a representative of RNA viruses, variation in the genome is favored by high mutation rates during the replication process, and the resulting genotypes are due to mutations and recombination. This results in the exchange of genetic material and the formation of new antigenic variants, which can escape immune pressure and lead to changes in cell tropism and host range [3-11].

The most variable protein of the foot-and-mouth disease virus is VP ₁ , which accounts for more than 90% of the mutations of all structural genes. The highest frequency of mutation processes is observed for 40-60, 130-160 and 190-213 aa. [12-16].

Isolates of foot-and-mouth disease virus serotype A are characterized by the greatest genetic diversity. Serotype A includes 3 characterized prototypes ASIA, AFRICA, EURO-SA and one still poorly studied group of isolates. The genetic and antigenic diversity of foot-and-mouth disease virus serotype A leads to problems in the specific prevention of foot-and-mouth disease when using culture-inactivated foot-and-mouth disease vaccines, and also complicates the strain-specific diagnosis of isolated foot-and-mouth disease virus isolates [17]. As a result, there is a need to create new diagnostic tools and specific immunoprophylaxis against foot-and-mouth disease virus serotype A and, in particular, the new genotype A/AFRICA/G-I, representatives of which have begun to spread throughout East Africa and beyond.

In East Africa, in particular in Kenya, Tanzania, and Ethiopia, outbreaks of foot-and-mouth disease of the A/AFRICA/G-I genotype are sporadic. With the strengthening of trade and economic ties with the countries of the African continent, in particular with East Africa, there are risks of introducing isolates of this genotype of the foot-and-mouth disease virus into the territory of the Russian Federation.

Analyzing the outbreaks that were recorded in Africa, it was found that in Kenya and Tanzania in the period from 2003 to 2013, isolates of the foot-and-mouth disease virus were identified that belong to the A/AFRICA/G-I genotype. The outbreaks were managed by applying serious control measures, however, in recent years, outbreaks of foot-and-mouth disease caused by a virus of the same genotype have again begun to be recorded in Ethiopia, Tanzania, and Kenya. This phenomenon seems dangerous and requires the study of strains of this genotype to create diagnostic tools and specific prevention of foot and mouth disease of the A/AFRICA/G-I genotype.

Production strains of foot-and-mouth disease virus serotype A are known, which are used for the production of specific foot-and-mouth disease prevention products:

- strain “A22/Iraq/64” (genotype A/ASIA/Iraq-64),

- strain “A/Türkiye/06” (genotype A/ASIA/Iran-05),

- strain “A/Krasnodar/2013” (genotype A/ASIA/Iran-05 ^SIS-10 ),

- strain “A/Zabaikalsky/2013” (genotype A/ASIA/Sea-97),

- strain “A/ARRIAH/2015” (genotype A/ASIA/G-VII),

- strain “A/KEN/42/66” (genotype A/AFRICA/G-I).

The foot-and-mouth disease virus isolate “A/TAN/5/2013” was isolated from cattle in the United Republic of Tanzania in 2013 and was supplied to the ARRIAH from the World Reference Laboratory for Foot-and-Mouth Disease, the Pirbright Institute, UK) for scientific research. As a result of this work, by adapting to cell cultures, the “A/Tanzania/2013” strain of foot-and-mouth disease virus was obtained.

According to the results of a comparative analysis of nucleotide sequences, the isolated isolate belongs to serotype A, topotype AFRICA, genetic line G-I of the foot-and-mouth disease virus, which differs significantly from the production strains of foot-and-mouth disease virus serotype A.

The foot and mouth disease virus strain “A/Tanzania/2013” has been deposited in the All-Russian State Collection of Exotic Types of Foot and Mouth Disease Viruses and Other Animal Pathogens (FMDVs) of the FGBU “ARRIAH” under registration number: No. 410 - dep / 22-44 - GKSHM FGBU “ARRIAH”.

The possibility of using the “A/Tanzania/2013” strain of foot-and-mouth disease virus for the production of diagnostic and preventative means for foot-and-mouth disease caused by the A/AFRICA/G-I genotype virus has been experimentally confirmed.

The phylogenetic tree was derived using the MEGA program and the Neighbor-Joining algorithm [18]. The percentage of replicate branches in which related taxa cluster together in a bootstrap test (100 replicates) is shown next to the branches [19]. Evolutionary distances were calculated using the Maximum Composite Likelihood method [20] and expressed in units of the number of base substitutions per site. This analysis included 32 nucleotide sequences. All items containing spaces and missing data were removed (complete deletion option). There were a total of 663 items in the final data set. Evolutionary analysis was carried out in MEGA X [21].

Considering the intensive trade relations between the Russian Federation and African countries, the likelihood of the risk of introducing foot-and-mouth disease into the territory of our country remains very high. Immunization is an effective tool in disease control. In connection with the emergence of a representative of this genotype of the foot-and-mouth disease virus and the need to protect animals from it, the Federal State Budgetary Institution "ARRIAH" developed a vaccine that includes the antigen of the foot-and-mouth disease virus of this strain.

There was a need to create a diagnostic test system to determine the titer of antibodies against the structural proteins of the foot-and-mouth disease virus strain “A/Tanzania/2013” using a liquid-phase enzyme-linked immunosorbent assay.

Protection against foot and mouth disease after vaccination is determined by the level of specific antibodies in the blood serum of animals [22-24]. The level of humoral immunity of vaccinated livestock is determined by identifying antibodies specific to the structural proteins of the foot-and-mouth disease virus. In accordance with the recommendations of the OIE (OIE) [2], the microneutralization reaction (MN) and enzyme-linked immunosorbent assay (ELISA) are used to determine the immune status of animals. The microneutralization reaction is considered sensitive and specific, however, the analysis has a number of the following limitations: 1) the long duration of the reaction (at least 72 hours), 2) the high cost of the procedure associated with the use of a sensitive cell line, the presence of a CO ₂ incubator and other equipment , 3) the use of non-inactivated foot-and-mouth disease virus, which is a biological agent of hazard group II and involves work only in BSL-3 level laboratories.

In turn, ELISA has many advantages, including rapidity, high specificity and sensitivity, suitability for large-scale screening of field samples and no requirement for special laboratory conditions, for example, cell culture or carbon dioxide environment, and does not involve working with high-level microorganisms danger [2]. Based on this, it is advisable to use ELISA to study the level of humoral immunity in vaccinated animals.

Depending on the phase in which the immune complexes are located, solid-phase and liquid-phase ELISA variants are distinguished. Particular attention should be paid to the liquid-phase blocking indirect “sandwich” version of ELISA, which is characterized by a number of advantages: 1) this analysis is as close as possible to the classical neutralization reaction, since the formation of the immune complex occurs in the liquid phase as the most natural conditions; 2) antigen epitopes are most accessible in space for specific antibodies, which allows increasing the sensitivity and specificity of the reaction.

Robiolo V., La Torre J., Duffy S., Leon E., Seki C, Torres A., Mattion N. (2010) developed a method for determining the titer of antibodies against foot-and-mouth disease virus antigens strains A ₂₄ /Cruzeiro, A/Argentina/01 , O1/Campos and C ₃ /Indaial [25]. Against these representatives of the virus, the reliability of determining the level of antibodies was at least 96%, while this test system made it possible to detect antibodies against the strain “A/Tanzania/2013” (genotype A/AFRICA/GI) with a diagnostic sensitivity of no more than 29% and specificity - no more than 50%.

Sharma G.K. et al. (2015) proposed a test system for detecting antibodies against foot-and-mouth disease virus types A, O, Asia-1 using a liquid-phase blocking indirect “sandwich” version of ELISA [26]. This technique made it possible to identify a wide range of anti-foot-and-mouth disease antibodies, but the specificity for the antigen of the foot-and-mouth disease virus genotype A/AFRICA/G-I was no more than 43%, and, therefore, accurate detection of immunoglobulins G was not possible. The authors of the test system used monoclonal antibodies, the use of which increased the range of detection of various genotypes of foot-and-mouth disease virus within one serotype, but at the same time the specificity with respect to the detection of antibodies against isolates/strains of a specific genotype, in particular “A/Tanzania/2013” (genotype A) was significantly reduced /AFRICA/G-I).

Considering the above, it is important to develop a test system for determining the titer of antibodies against the structural proteins of the foot and mouth disease virus strain “A/Tanzania/2013” using a liquid-phase enzyme-linked immunosorbent assay.

The above test systems are not presented on the market as kits for the detection of foot-and-mouth disease antibodies in animal blood sera and remain unavailable to the general consumer. In turn, the world market currently has European kits for detecting antibodies against foot-and-mouth disease virus type A - IZSLER (Italy) and PrioCHECK (Netherlands).

The test system in the IZSLER kit is intended for the detection of anti-foot-and-mouth disease antibodies using a solid-phase direct “sandwich” ELISA version [27]. The advantage of this kit is the use of monoclonal antibodies, however, they were obtained before 2013 (the period when the production strain “A/Tanzania/2013” appeared) and cannot detect specific antibodies in animal blood sera with high reliability and accuracy. The specificity and sensitivity of this test system for detecting antibodies against foot-and-mouth disease virus genotype A/AFRICA/G-I is below 44%, which is confirmed by microneutralization reaction data in a sensitive continuous monolayer pig kidney cell line IB-RS-2. In other words, the test system used does not allow obtaining reliable results specifically for isolates and strains of foot-and-mouth disease virus genotype A/AFRICA/G-I.

The closest prototype for the test system we developed is the PrioCHECK test system (Prionics Lelystad B.V., Netherlands) for detecting antibodies to foot-and-mouth disease virus type A [28].

The components for the corresponding kit are presented below:

Component 1 - Immunological tablets.

Component 2 - Conjugate (30x). The diluted conjugate is not stable; prepare immediately before use.

Component 3 - Dilution Buffer (5x).

Component 4 - Non-immune horse blood serum.

Component 5 - Demineralized water.

Component 6 - Wash buffer (200x).

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

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

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

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

Component 11 - Chromogen substrate (TMB).

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

This test system includes a conjugate, which is a labeled monoclonal antibody against foot-and-mouth disease virus serotype A, and therefore is type-specific, but without a narrower differentiation. The prototype test system was developed back in the late 90s of the 20th century. It uses antibodies against foot-and-mouth disease virus antigens of various genotypes of type A, except for the newly emerged ones, in particular A/AFRICA/GI. Isolates and strains of this line have significant differences from other lines of serotype A. Analyzing the nucleotide and amino acid sequence of the highly variable VP ₁ protein of the foot-and-mouth disease virus, scientists came to the conclusion that there are a sufficient number of significant substitutions to classify the virus as a different genotype. Based on the above, the prototype test system is not highly sensitive and specific to the foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/GI. This test system detects antibodies to this strain by no more than 41%.

Today, using a liquid-phase blocking “sandwich” version of the ELISA with the characteristics of the reaction and a certain composition of the reaction components, a “Test system based on a liquid-phase blocking indirect “sandwich” version of the ELISA has been developed to determine the titer of antibodies against the foot-and-mouth disease virus strain A No. 2269/ ARRIAH/2015 genotype A/ASIA/G-VII in animal blood sera after immunization”, “Test system based on liquid-phase blocking indirect “sandwich” ELISA variant for determining antibody titer against foot-and-mouth disease virus strain O No. 2047/Saudi Arabia/2008 genotype O/ME-SA/PanAsia2 in animal blood sera after immunization" and "Method for rapid quantitative determination of the level of humoral immunity of animals to strain O No. 2311/Zabaikalsky/2016 genotype O/ME-SA/Ind-2001 of foot-and-mouth disease virus after vaccination with liquid-phase “sandwich” version of ELISA” [29-31].

Based on this, it is necessary to develop a sensitive, specific test system for determining the titer of antibodies against the structural proteins of the foot-and-mouth disease virus strain “A/Tanzania/2013” using a liquid-phase enzyme-linked immunosorbent assay with certain features in the analysis and component composition.

The prototype test system is based on a solid-phase competitive “sandwich” version of the ELISA, however, this form is significantly removed from the microneutralization reaction in terms of its ability to capture antigenic sites compared to the liquid-phase blocking indirect “sandwich” version of the ELISA.

The prototype version assumes the presence in its composition of control animal blood sera in liquid native form, which does not allow this component to be stored for a long time as part of the test system.

The prototype version also has some limitations in application, namely, the working solution of the conjugate is not stable and quickly deteriorates. As part of the kit, it is presented in the form of a 30-fold concentrate. In this state, unlike the lyophilized component, the shelf life is noticeably 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 densities and the level of humoral immunity against the foot-and-mouth disease virus antigen.

The prototype test system uses a direct conjugate, which is convenient to use, but this increases the likelihood of background noise phenomena, which can negatively affect the final results of a quantitative study of animal blood sera.

Considering the found limitations in the use of existing test systems, including the prototype, for carrying out an accurate quantitative analysis of the level of humoral immunity of animals after inoculation with foot-and-mouth disease vaccines containing inactivated foot-and-mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I, it is relevant development of a test system for determining the titer of antibodies against the structural proteins of the foot-and-mouth disease virus strain “A/Tanzania/2013” using a liquid-phase enzyme-linked immunosorbent assay, taking into account the above disadvantages.

Currently, the veterinary service of the Russian Federation does not have a modern domestic test system that would allow high reliability to quantify the titer of antibodies against the structural proteins of the foot-and-mouth disease virus strain “A/Tanzania/2013” using ELISA.

The purpose of the invention is to create a test system for determining the antibody titer against the structural proteins of the foot-and-mouth disease virus strain “A/Tanzania/2013” using a liquid-phase enzyme-linked immunosorbent assay.

This goal is achieved by the fact that the proposed test system contains the following freeze-dried immunospecific components with a high degree of activity:

1) cultural inactivated lyophilized foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I;

2) sensitizing antibodies - rabbit immunoglobulins G against the structural proteins of the foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I;

3) detector antibodies - guinea pig immunoglobulins G against the structural proteins of the foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I;

4) control 1 (positive control) - positive calf blood serum to the structural proteins of the foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I with an inhibition percentage of 75-100%;

5) control 2 (positive control) - positive calf blood serum to the structural proteins of the foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I with a percentage of inhibition from 50.0% to 74.9%;

6) control 3 (negative control) - calf blood serum that does not contain antibodies to the foot-and-mouth disease virus antigen;

7) blocking component;

8) anti-species immunoperoxidase conjugate - rabbit immunoglobulins against guinea pig Ig G, conjugated with horseradish peroxidase.

The proposed test system also contains nonspecific components: a buffer solution based on sodium carbonate and sodium bicarbonate (pH 9.5-9.7); 20x buffer solution concentrate; chromogenic substrate - ABTS; “stop” solution - 1.0% sodium cocosulfate solution.

The goal is achieved due to the fact that the liquid-phase blocking indirect “sandwich” version of ELISA is closest to the classical microneutralization reaction in IB-RS-2 cell culture, since all antigenic sites of the structural proteins of the foot-and-mouth disease virus strain “A/Tanzania/2013” are genotype A/ AFRICA/G-I are free in the liquid phase and are open to binding to specific sensitizing and detector antibodies. The reaction is based on the interaction of immunoglobulins G of the animal blood serum under study and inactivated foot-and-mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I in the liquid phase with the formation of immune complexes. In parallel with this, the wells of the flat-bottomed plate are sensitized with rabbit antibodies against the foot-and-mouth disease virus of the same genotype. After this, the sensitized wells are treated with blocking blood serum, the resulting immune complexes from a round-bottom plate are added, incubated, and a suspension of detector antibodies restored from a lyophilized dried state is added. The resulting complex immune complex is detected using antibodies against guinea pig immunoglobulin G conjugated to horseradish peroxidase and a chromogenic substrate.

The technical result of the invention consists in the development of a modern, specific against the structural proteins of the foot-and-mouth disease virus strain "A/Tanzania/2013" genotype A/AFRICA/G-I and a sensitive test system designed to determine the titer of antibodies against the structural proteins of the specified strain of foot-and-mouth disease virus using the liquid-phase blocking method indirect “sandwich” version of ELISA based on the use of inactivated foot-and-mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I and when obtaining immunospecific reaction components.

Unlike the prototype, the developed test system uses a culture-concentrated 700-fold inactivated foot-and-mouth disease virus strain “A/Tanzania/2013”, which is presented in the form of a freeze-dried suspension. This fact makes it possible to quantify humoral immunity against the foot-and-mouth disease virus of the specified strain with high rates of sensitivity (more than 99%) and specificity (100%), which is 59% higher than the closest prototype. In addition, the lyophilized component remains stable for at least 3 years compared to the antigen in its native state, which is an advantage in using this test system. The activity of the obtained antigen of foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I is 1:2500.

Unlike the prototype, sensitizing and detector antibodies of rabbit and guinea pig are used, respectively, highly specific to the structural proteins of the foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I. The rabbit and guinea pig immunoglobulins G indicated in the ELISA test system against the specified strain of foot-and-mouth disease virus are presented in the form of a lyophilic component, which allows immunoglobulins G to remain in a stable state for at least 3 years with an activity of at least 1:10000 and 1:5000, respectively.

For control 1, the activity is at least 1:3500, for control 2 - at least 1:4000. This activity is convenient for obtaining a large volume of controls in a working native state for direct ELISA.

Unlike the prototype version, the presented test system provides for control of the antigen and conjugate, which makes it possible to take into account background values and, thereby, obtain reliable data on optical density values and antibody titer against the structural proteins of the foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I. The activity of the conjugate is 1:2500.

The developed test system is based on the specific blocking of the antigen (structural proteins) of the foot-and-mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I (Fig. 3) by antibodies contained in the test serum sample in the liquid phase. The “test serum/antigen” mixture is transferred into the wells of a plate previously immobilized with rabbit sensitizing antibodies against the “A/Tanzania/2013” strain of genotype A/AFRICA/G-I.

The presence of antibodies to the foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I in the test sample will be expressed in the formation of an immune complex and a decrease in the amount of free antigen that is bound by sensitizing antibodies. The antigen fixed in the wells interacts with guinea pig detector antibodies, which are detected by reaction with an immunoperoxidase conjugate against guinea pig Ig G and subsequent staining with a chromogenic substrate. The intensity of staining is inversely proportional to the quantitative value of the detected level of humoral immunity of the animal. The color intensity resulting from the reaction is taken into account using a spectrophotometer.

The essence of the invention is reflected in graphic images: Fig. 1 - Sedimentation profile of antigen fractions of the foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I.

Fig. 2 - Electropherogram for the antigen of the foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I in 12% polyacrylamide gel. Note: 1 - fraction 146S of the component purified for immunization of animals and obtaining the antigen of the virus of a given strain, M - molecular weight marker.

Fig. 3 - Phylogenetic affiliation of the strain “A/Tanzania/2013” genotype A/AFRICA/G-I.

The essence of the invention is explained in the list of sequences, in which:

SEQ ID NO:l represents the nucleotide sequence of the ID gene encoding the VP1 protein of the A/Tanzania/2013 strain of genotype A/AFRICA/G-I foot-and-mouth disease virus;

SEQ ID NO:2 represents the amino acid sequence of the VP ₁ protein of the A/Tanzania/2013 strain of genotype A/AFRICA/GI foot and mouth disease virus.

At the preparatory stage of the study, lyophilized immunospecific components of the test system (antigen (structural proteins) of the foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I, immunoperoxidase conjugate, positive and negative controls, sensitizing and detector antibodies) are restored to the required volume deionized water.

Working solutions are prepared.

Solution No. 1 (saline buffer solution of sodium carbonate and sodium bicarbonate). Prepare 50 ml of a solution with a pH value of 9.5-9.7 using standard methods.

Solution No. 2 (Washing solution). Prepare 2 liters of standard TBS buffer solution with the addition of 0.1% tween-20 with a pH value of 7.5-7.7. Working solution No. 2 is prepared by diluting the concentrate 20 times.

Solution No. 3 (buffer for the recovery of antibodies and conjugate). The working buffer solution for diluting antibodies and immunoperoxidase conjugate is prepared immediately before use from working solution No. 2 with the addition of a 10% solution of the blocking component (fetal calf serum).

The stages of conducting a liquid-phase blocking indirect “sandwich” version of the ELISA to determine the titer of antibodies against the structural proteins of the foot-and-mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I are presented below.

1. Immobilization of rabbit antibodies against the structural proteins of the foot and mouth disease virus. A restored suspension of sensitizing antibodies against the structural proteins of the foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I is prepared at a dilution of 1:10,000 in a saline buffer solution of sodium carbonate and sodium bicarbonate (solution No. 1). 100 μl of a suspension of these antibodies is added to all wells of an immunological 96-well plate, covered with a lid and incubated for 18-20 hours at a temperature of (2-8)°C.

2. The wells of the plate are washed using solution No. 2 twice.

3. Formation of an immune complex in the liquid phase. In parallel with the immobilization of the tablet, a serological reaction of antigen binding is carried out with test and control samples of animal blood serum. The following dilutions of animal blood serum are prepared: 4.0; 5.0; 5.5; 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; 9.0; 9.5; 10.0; 10.5; 11.0 log ₂ SN ₅₀ (1:16-1:2048) in solution No. 2. To do this, 0.047 cm ³ (47 μl) of a buffer solution and 0.003 cm ³ (3 μl) of a sample are added to all wells of the plate with the exception of wells H1-6, which are left for antigen control (CA) and conjugate control (CC). Add 0.05 cm ³ (50 μl) of buffer solution to wells H1-6. Test and control samples are placed on the plate according to Table 1. The serum is considered positive when the level of antibodies against the structural proteins of the foot-and-mouth disease virus is ≥5.0 log ₂ SN ₅₀ (≥1:32).

As control 1, positive blood serum of cattle/pigs is used against the structural proteins of the foot and mouth disease virus strain "A/Tanzania/2013" genotype A/AFRICA/G-I - percentage of inhibition (PI) 75-100%, control 2 - positive blood serum of cattle/pigs against structural proteins of foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I -PI=50.0-74.9%, control 3 - normal cattle/pig serum - percentage of inhibition <50%.

Add 0.05 cm ³ of the working dilution of the antigen in solution No. 2 to all wells of the plate in accordance with a dilution of 1:2500. After mixing the contents of the wells, the plate is carried out for 1 hour at a temperature of (2-8)°C.

4. Antigen capture. 0.05 cm ³ of a mixture of control and test samples and antigen is added to the washed wells of the immobilized plate, the plate is closed with a lid and incubated for 60±2 minutes at a temperature of (37±0.2)°C.

5. Washing the wells of the plate. Carry out as described above (item 2). The procedure is repeated 3 times.

6. Addition of detector antibodies. In all wells of the plate, with the exception of wells with QC (conjugate control), where 0.05 cm ³ of solution No. 3 is added, 0.05 cm ³ of the working dilution of detector antibodies (1:5000) in solution No. 3 is added. The plate is covered with a lid and incubated for 60°2 minutes at a temperature of (3±0.2)°C.

7. Washing the wells of the plate. Carry out as described above (item 2). The procedure is repeated 3 times.

8. Addition of the conjugate. Add 0.05 cm ³ of the working dilution of the conjugate (1:2500) in solution No. 3 to all wells of the plate. The plate is covered with a lid and incubated for 60±2 minutes at a temperature of (37±0.2)°C.

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

10. Manifestation of color reaction. Add 0.05 cm ³ of ABTS chromogenic substrate to all wells of the plate, cover with a lid and incubate for 20 minutes at a temperature of (20±0.2)°C.

11. Inhibition of the reaction. The reaction is stopped by adding 0.05 cm ^{3 of} 1% sodium coco sulfate solution to each well of the plate.

12. Determination of the absorption value and the level of humoral immunity according to ELISA data. The results of the analysis are taken into account in an instrumental way. Immediately after stopping the reaction, measure the absorbance value (A) of the reaction products in each well at a wavelength of 415 nm using a microplate spectrophotometer with a vertical light beam. The A values are converted to the percentage of inhibition (PI, %) using the formula:

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

where, A _serum is the average absorption value of a mixture of serum with inactivated foot-and-mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/GI;

A _ka is the average absorption value of the antigen control;

And _kk is the average value of the absorptivity of the conjugate control.

The reliability of the analysis performed using the proposed test system is determined if the following criteria are met:

- the difference between the values of A _ka and A _kk is at least 0.4 optical units (p.u.);

- control 1 has a PI value>75.0%;

- control 2 has a PI value in the range of 50.0-74.9%;

- control 3 has a value of PI<50.0%.

Blood sera for which the value is ≥50% are considered positive, and the animals from which they were obtained are considered immune to foot and mouth disease caused by the virus strain “A/Tanzania/2013” genotype A/AFRICA/GI. The PI value≤50% in the blood serum of the examined animals is negative, which indicates the absence of specific antibodies to the structural proteins of the foot-and-mouth disease virus “A/Tanzania/2013” genotype A/AFRICA/GI. 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 virus strain “A/Tanzania/2013” genotype A/AFRICA/GI and is expressed in log ₂ SN ₅₀ .

As a result of the experiments, the original components of the reaction were titrated. The activity of detector antibodies in ELISA was 1:5000, sensitizing antibodies - 1:10000, anti-species conjugate - 1:2500; foot and mouth disease virus antigen strain "A/Tanzania/2013" genotype A/AFRICA/G-I - 1:2500, control 1 - 1:3500, control 2 - 1:4000. Using the developed test system, a study of animal blood serum samples was carried out with a quantitative determination of the level of humoral immunity and a comparative analysis was carried out with the results of RMN in a sensitive continuous monolayer pig kidney cell line (IB-RS-2) [2].

The developed test system makes it possible to simultaneously test 6 serum samples in the following dilutions in the format of one 96-well plate: 4.0; 5.0; 5.5; 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; 9.0: 9.5; 10.0; 10.5; 11.0 log ₂ SN ₅₀ (1:16-1:2048).

For the work, we used the inactivated antigen of the foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I. This strain is currently used for the development of serological test systems exclusively in the Russian Federation.

Monitoring the strain “A/Tanzania/2013” genotype A/AFRICA/G-I of foot-and-mouth disease virus for specificity.

The viral isolate that served as the source for obtaining the A/Tanzania/2013 strain of genotype A/AFRICA/GI of foot-and-mouth disease virus was isolated from cattle in the United Republic of Tanzania. Specificity was determined using RT-PCR followed by sequencing of the ID gene corresponding to the VP ₁ protein, in which mutations most often occur. Based on the results of a study of the nucleotide sequence of this gene, it was revealed that the strain “A/Tanzania/2013” belongs to the genotype A/AFRICA/GI. The calculation was carried out in accordance with Bayesian probability theory. Specificity was also confirmed in the complement fixation reaction with strain-specific serum. The concentration of the total viral protein in the studied antigen of the foot-and-mouth disease virus strain “A/Tanzania/2013” was 3.02 μg/ml, the concentration of the 146S component was 2.05 μg/ml, the 146+75S component was 2.90 μg/ml, 12S particles - 0.12 mcg/ml. The possibility of using the “A/Tanzania/2013” strain of foot-and-mouth disease virus for the development of diagnostic test systems has been experimentally confirmed.

Morphological characteristics

Strain “A/Tanzania/2013” of foot-and-mouth disease virus serotype A belongs to the family Picornaviridae, genus Aphthovirus, species Foot-and-Mouth Disease Virus and has morphological characteristics characteristic of the causative agent of foot-and-mouth disease: the shape of the virion is ixahedral, size 25 nm. A virion consists of a single-stranded, positive-sense RNA molecule enclosed in a protein shell. It consists of 32 capsomeres arranged in cubic symmetry.

Antigenic properties

According to its antigenic properties, the strain “A/Tanzania/2013” of the foot-and-mouth disease virus belongs to serotype A. The virus is stably neutralized by a homologous antiserum. The virus does not exhibit hemagglutinating activity. In recovered animals, type-specific antibodies are formed in the blood serum, which are detected in an enzyme-linked immunosorbent assay and microneutralization reaction (RMN).

Using the method of nucleotide sequencing, the primary structure of the ID gene of the VP ₁ protein of the “A/Tanzania/2013” strain of foot-and-mouth disease virus was determined. Comparative analysis of nucleotide sequences showed that the “A/Tanzania/2013” strain of foot-and-mouth disease virus belongs to the A/AFRICA/GI genotype (Fig. 3).

The antigenic affinity (r ₁ ) of the “A/Tanzania/2013” strain of foot-and-mouth disease virus was studied in a cross-study of the strain with specific sera obtained for the following production strains of foot-and-mouth disease virus:

- strain “A ₂₂ /Iraq/64” (genotype A/ASIA/Iraq-64),

- strain “A/Türkiye/06” (genotype A/ASIA/Iran-05),

- strain “A/Krasnodar/2013” (genotype A/ASIA/Iran-05 ^S1S-10 ),

- strain “A/Zabaikalsky/2013” (genotype A/ASIA/Sea-97),

- strain “A/ARRIAH/2015” (genotype A/ASIA/G-VII),

- strain “A/KEN/42/66” (genotype A/AFRICA/G-I) in RMN.

The titer of reference cattle blood sera obtained by immunizing animals with monovalent vaccines from production strains of foot-and-mouth disease virus serotype A against 10 ² TCD ₅₀ of homologous and heterologous virus was determined in RMN with cross-titration, calculating values using a linear regression equation, and expressed in lg. The r ₁ value was determined as the antilogarithm of the difference in serum lg titers against heterologous and homologous viruses [7, 8, 9].

The value of r ₁ in the RMN was interpreted as follows:

at ≥0.3 - the studied and production strains of foot-and-mouth disease virus are closely related;

at <0.3 - the studied sample of the foot-and-mouth disease virus strain differs from the production strain.

Indicators of antigenic relatedness when studying the strain “A/Tanzania/2013” amounted to r ₁ from 0.03 to 0.20, namely, for the strain “A ₂₂ /Iraq/64” - 0.11, for the strain “A/Turkey/ 06" - 0.03, for strain "A/Krasnodar/2013" - 0.05, for strain "A/Zabaikalsky/2013" - 0.09, for strain "A/ARNIAH/2015" - 0.14, for strain “A/KEN/42/66” - 0.20, which indicates the absence of antigenic relationship with production strains of foot-and-mouth disease virus serotype A. Geno- and chemotaxonomic characteristics

Strain “A/Tanzania/2013” of foot-and-mouth disease virus is an RNA (+)-containing virus with a molecular weight of 7.01X10 ⁶ D. Nucleic acid is represented by a single-chain linear molecule with a molecular weight of 2.85 × 10 ⁶ D. The virion has a protein shell consisting only of four proteins VP ₁ , VP ₂ , VP ₃ and VP ₄ . The following main non-structural functional proteins of the foot-and-mouth disease virus are distinguished: 2A, 2B, 2C, 3A, _{3B1 3B2} , _3B3 , 3C, 3D (RNA-dependent RNA polymerase).

The main antigenic protein is VP ₁ . The virion contains approximately 31.5% RNA and 68.5% protein. Viral RNA (+) is infectious and is involved in the formation of precursor proteins in infected cells.

Physical properties

The mass of the virion is 8.43×10 ^-18 g. The buoyant density is 1.43 g/cm ³ .

Resistance to external factors

Strain “A/Tanzania/2013” of foot and mouth disease virus is resistant to ether, chloroform, and acetone. Most stable at pH 7.45-7.65. Shifts in pH to the acidic and strongly alkaline side lead to inactivation of the virus. Sensitive to formaldehyde, UV irradiation, γ-irradiation, high temperatures (above 37.8°C).

Additional characteristics and properties

Reactogenicity - does not have reactogenic properties.

Pathogenicity - pathogenic for artiodactyl animals.

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

Stability - retains the original biological properties when passaging in sensitive biological systems for 5 passages (observation period) on continuous crops.

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

Example 1. Obtaining the antigen of the foot and mouth disease virus strain “A/Tanzania/2013”.

To obtain the antigen of the foot-and-mouth disease virus strain “A/Tanzania/2013” for diagnostic purposes, suspension cultivation of the virus was carried out in the cell line BHK-21/SUSP/ARRIAH for 9-11 hours to obtain a viral suspension with an infectious activity titer of at least 6.5 lg TCD50/SM ³ . The resulting viral suspension was inactivated with aminoethylethylenimine and clarified with polyhexamethylene guanidine.

The inactivated cultural suspension containing the foot-and-mouth disease virus antigen was clarified for 30 minutes. at 6000 rpm and 4±0.2°C. 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%, mixed vigorously and kept at a temperature of 4±2°C for 18-24 hours. The viral suspension was precipitated at 6000 rpm for 60 minutes, the sediment was dissolved in a 1/15 M solution of phosphate-buffered saline, concentrating 700 times (1/700 of the original volume).

50% chloroform was added to the resulting precipitate, mixed vigorously and fractionated using a centrifuge for 30 minutes. at 3000 rpm. The upper aqueous fraction containing the antigen of the foot-and-mouth disease virus strain “A/Tanzania/2013” was selected. 100 μl of sample was taken for subsequent electrophoretic analysis in a 12% polyacrylamide gel under denaturing conditions.

At the next stage, 146S particles of the foot-and-mouth disease virus strain “A/Tanzania/2013” were obtained (structural proteins of the foot-and-mouth disease virus, this component is immunogenic and the most important in the immunization of animals).

To isolate complete particles with a sedimentation coefficient of 146S, a linear gradient of sucrose was prepared with concentrations of 50, 40, 30, 20, and 10% (in the direction of the layers in the centrifuge tube from bottom to top). A suspension of the antigen of the foot-and-mouth disease virus strain “A/Tanzania/2013” was added in 15 ml quantities to centrifuge tubes, then sucrose solutions were successively layered, starting with a 10% solution and ending with a 50% solution. The tubes were placed in metal centrifuge beakers and, after careful equilibration, were centrifuged at a speed of 30,000 rpm and a temperature of 4±0.2°C for 4 hours. Fractionation of the sucrose gradient was carried out using a peristaltic pump, collecting 1.0 ml fractions into separate tubes.

The 146S component was reprecipitated by ultracentrifugation for 4 hours at 25,000 rpm. and a temperature of 4±0.2°C and resuspended the sediment in 1/15 M phosphate-buffered saline solution.

The results of a spectrometric study of the obtained antigen fractions of the foot and mouth disease virus strain “A/Tanzania/2013” in the form of an antigenic profile are presented in Fig. 1. Electrophoresis of the purified 146S component fraction was carried out in a 12% polyacrylamide gel under denaturing conditions (Fig. 2).

Example 2. Preparation of hyperimmune rabbit blood serum (sensitizing antibodies) against the structural proteins of the foot and mouth disease virus strain “A/Tanzania/2013”.

To obtain specific blood sera against the structural proteins of the foot-and-mouth disease virus strain “A/Tanzania/2013,” clinically healthy rabbits of average fatness weighing 2.5-3.0 kg were used.

For hyperimmunization of animals, a concentrated purified inactivated antigen of the foot and mouth disease virus strain “A/Tanzania/2013” was used, obtained as described in example 1, from which an emulsion was prepared using the oil adjuvant Montanide ISA-61 VG (in the adjuvant/antigen ratio = 60/40 mass). The resulting vaccine was injected into the muscle of the hind limbs of a rabbit on days 0, 14 and 28 in a volume of 0.5 cm ³ . 7 days after the last immunization, blood containing sensitizing antibodies was collected from rabbits, 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 the structural proteins of the foot and mouth disease virus strain “A/TANZANIA/2013”.

For hyperimmunization of guinea pigs, an emulsion of a purified preparation of concentrated inactivated foot-and-mouth disease virus strain “A/Tanzania/2013” was used, obtained as described in example 1. The preparation of the vaccine and the immunization scheme were the same as in example 2.

Example 4. Determination of the activity in ELISA of the antigen of the foot-and-mouth disease virus strain “A/Tanzania/2013”, sensitizing and detector antibodies to structural proteins.

Animal blood serum samples were tested for activity and specificity in a liquid-phase blocking indirect “sandwich” version of the ELISA. A selection of optimal working dilutions of these components was carried out to obtain reliable results in ELISA. The obtained analysis data are presented in tables 2, 3, 4.

For analysis, the following dilutions of sensitizing antibodies were studied: 1:8000, 1:9000, 1:10000, 1:11000. The dilutions of the remaining components were constant: for antigen - 1:2500, for detector antibodies - 1:5000, conjugate - 1:2500. Sera specific to certain strains of foot-and-mouth disease virus with an activity of 8.0 log ₂ SN ₅₀ were used as control sera for analysis. From table 2 it follows that with a dilution of sensitizing antibodies of 1:10000, a reliable result was achieved (1:256 or 8.0 log ₂ SN ₅₀ ). At lower dilutions of antibodies, high background values were observed, which did not allow obtaining a correct result. At higher dilutions, a decrease in the detectable values of the level of humoral immunity in the blood serum of animals was noted. When studying nonspecific and negative sera, negative results were obtained, which indicated the high specificity of the developed method.

The following dilutions of detector antibodies were used for analysis: 1:3000, 1:4000, 1:5000, 1:6000. The dilutions of the remaining components were constant: for antigen - 1:2500, for sensitizing antibodies - 1:10000, conjugate - 1:2500. Sera specific to certain strains of foot-and-mouth disease virus with an activity of 8.0 log ₂ SN ₅₀ were used as control sera for analysis. From table 3 it follows that with a dilution of detector antibodies of 1:5000, a reliable result was achieved (1:256 or 8.0 log ₂ SN ₅₀ ). At lower dilutions of antibodies, high background values were observed, which did not allow obtaining a correct result. At higher dilutions, a decrease in the detectable value of antibody titer in animal blood sera was observed. When studying nonspecific and negative sera, negative results were noted, which indicated the high specificity of the developed method.

The following antigen dilutions were tested for analysis: 1:1500, 1:2000, 1:2500, 1:3000. The dilutions of the remaining components were constant: for detector antibodies - 1:5000, for sensitizing antibodies - 1:10000, conjugate - 1:2500. Sera specific to certain strains of foot-and-mouth disease virus with an activity of 8.0 log ₂ SN ₅₀ were used as control sera for analysis. From table 4 it follows that with a dilution of the antigen of 1:2500, a reliable result was achieved (1:256 or 8.0 log ₂ SN ₅₀ ). At lower dilutions, high background values were observed, which did not allow obtaining a correct result. At higher dilutions, a decrease in the detectable value of antibody titer in animal blood sera was observed.

When examining nonspecific and negative sera, negative results were noted, which indicated the high specificity of the developed test system.

Thus, in the course of laboratory studies it was proven that the working dilutions of the antigen of the foot and mouth disease virus strain “A/Tanzania/2013”, sensitizing and detector antibodies were 1:2500, 1:10000, 1:5000, respectively.

Example 5. Use of a test system to determine the titer of antibodies against the structural proteins of the foot-and-mouth disease virus strain “A/Tanzania/2013” using liquid-phase ELISA

We studied 12 animal blood sera obtained after immunization with inactivated sorbed vaccines against foot-and-mouth disease containing the antigen of the foot-and-mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I. The values of the level of humoral immunity were determined using the developed ELISA test system (the proposed invention), and these sera were examined in the microneutralization reaction (RMN) in the sensitive continuous monolayer cell line IB-RS-2, recommended by the OIE (OIE) [2]. The results obtained are reflected in Table 5, from which it follows that the degree of correlation between the results of the developed ELISA test system (the proposed invention) and the classical RMN method is high (close to 100%).

Example 6. Determination of diagnostic indicators of the developed test system for determining the titer of antibodies against the structural proteins of the foot-and-mouth disease virus strain “A/Tanzania/2013” using a liquid-phase enzyme-linked immunosorbent assay (the proposed invention).

The presented test system for determining the antibody titer against the structural proteins of the foot-and-mouth disease virus strain “A/Tanzania/2013” using a liquid-phase enzyme-linked immunosorbent assay (the proposed invention) was examined for standard diagnostic indicators. To determine the sensitivity of the proposed test system, 450 animal blood sera were analyzed, which were obviously positive according to the microneutralization reaction in the sensitive continuous monolayer pig kidney cell line IB-RS-2. The antibody level for these blood sera was in the range of 5.5-10.0 log ₂ SN ₅₀ . ELISA was performed as described above. The developed test system (the proposed invention) determined that out of 450 blood serum samples, 448 were identified as positive, and 2 as negative (the level of humoral immunity according to ELISA was 1:24).

To study the specificity of the method, 185 negative animal blood sera were tested. As a result of the study using ELISA (the proposed invention), it was determined that all 185 were determined to be negative.

It was determined that in the 95% confidence interval, diagnostic sensitivity (DSe) was 98.41-99.95%, diagnostic specificity (DSp) - 98.03-100.00%, k-criterion - 0.992; negative predictive value (NPV) - 96.19-99.87%, overall accuracy (DAc) - 98.87-99.96% (Table 6).

Thus, the proposed “Test system for determining the titer of antibodies against the structural proteins of the foot-and-mouth disease virus strain “A/Tanzania/2013” using a liquid-phase enzyme-linked immunosorbent assay” is specific and sensitive, allowing the analysis of animal blood sera with a quantitative determination of the titer of antibodies against the structural proteins of the virus foot and mouth disease strain “A/Tanzania/2013” genotype A/AFRICA-I. This strain and its corresponding genotype are current and rapidly spreading throughout the African continent, as well as in the countries of the Middle East. The developed test system will be aimed at confirming the high effectiveness of inactivated foot-and-mouth disease vaccines produced in the Russian Federation containing the antigen of this strain, and thereby protecting the territory of Russia and the countries bordering it in the south from the spread of the foot-and-mouth disease virus genotype A/AFRICA/G-I.

Sources of information taken into account when compiling a description of the invention for the application for a RF patent for the invention “Test system for determining the titer of antibodies against the structural proteins of the foot-and-mouth disease virus strain “A/Tanzania/2013” using liquid-phase enzyme-linked immunosorbent assay”:

1. Gruzdev, K.N. Program of joint actions for the prevention and control of foot and mouth disease in animals in the CIS countries / K.N. Gruzdev, V.M. Zakharov, A.M. Rakhmanov // Proceedings of the Federal Center for Animal Health. -Vladimir, 2005. - T. 3. - P.3-13.

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. - P. 31-41.

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

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22. Oem J.K., Park J.FL, Lee K.N., Kim Y.J., Kye S.J., Park J.Y., Song H.J. Characterization of recombinant foot-and-mouth disease virus pentamer-like structures expressed by baculovirus and their use as diagnostic antigens in a blocking ELISA // Vaccine. - 2007. - V. 25(20). - P. 4112-412.

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24. 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.

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Claims (4)

1. Test system for determining the titer of antibodies against the structural proteins of the foot-and-mouth disease virus strain “A/Tanzania/2013” using a liquid-phase enzyme-linked immunosorbent assay, containing: - immunospecific components: foot and mouth disease virus antigen strain “A/Tanzania/2013” genotype A/AFRICA/G-I, lyophilized with ELISA activity 1:2500; sensitizing antibodies - rabbit immunoglobulins G against the structural proteins of the foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I, lyophilized with ELISA activity 1:10000; detector antibodies - guinea pig immunoglobulins G against the structural proteins of the foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I, lyophilized with ELISA activity 1:5000; control 1 - positive calf blood serum to the structural proteins of the foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I, lyophilized with an inhibition percentage of 75-100%; control 2 - positive calf blood serum to the structural proteins of the foot and mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I, lyophilized with a percentage of inhibition from 50% to 74.9%; control 3 - negative calf blood serum that does not contain antibodies to the foot-and-mouth disease virus; blocking component; anti-species immunoperoxidase conjugate - rabbit immunoglobulins against guinea pig Ig G, conjugated with horseradish peroxidase, with ELISA activity 1:2500; - non-specific components: buffer solution based on sodium carbonate and sodium bicarbonate with pH 9.5-9.7; 20x buffer solution concentrate; chromogenic substrate - ABTS, “stop” solution - 1.0% sodium coco sulfate solution; characterized in that for the production of immunospecific components, a culturally inactivated foot-and-mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/G-I is used; and to determine the percentage of inhibition, absorption data are used, while the titer of antibodies against the structural proteins of the foot-and-mouth disease virus strain “A/Tanzania/2013” is defined as the maximum dilution of the test material, for which the percentage of inhibition is ≥50%. 2. The test system according to claim 1, characterized in that it is specific and sensitive, allowing you to determine the titer of antibodies against the structural proteins of the foot-and-mouth disease virus strain “A/Tanzania/2013” genotype A/AFRICA/GI within 3 hours and quantify the level humoral immunity in a wide range of 4.0-11.0 log ₂ SN ₅₀ .