Use of sandwich blocking elisa reagent in preparation of foot-and-mouth disease virus antibody detection kit

Abstract

The application relates to application of a sandwich blocking ELISA reagent in preparation of a foot-and-mouth disease virus antibody detection kit, and belongs to the technical field of biology. Negative and positive sera and the detected serum are directly diluted on an ELISA plate coated with a capture antibody, then enzyme-labeled antibodies are added, finally virus antigens are added, and a virus antigen control is set. The foot-and-mouth disease type specific antibodies in the detected serum and the negative and positive sera block the double-antibody sandwich ELISA reaction of the virus antigens and the enzyme-labeled monoclonal antibodies with the capture antibodies on the ELISA plate, the blocking ELISA is judged, 50% of the average value of the OD values of the virus antigen control holes is taken as the critical value of 50% blocking of the virus antigens, the blocking sandwich ELISA method is successfully established, also called one-step liquid-phase blocking ELISA, and can be used for qualitative and quantitative detection of foot-and-mouth disease antibodies, and provides new technical support for foot-and-mouth disease vaccine immune antibody monitoring and vaccine immune effect evaluation.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a new method (sandwich blocking ELISA) for detecting antibodies against foot-and-mouth disease virus. Background Technology

[0002] Foot-and-mouth disease (FMD) is an acute, febrile, and highly contagious disease of cloven-hoofed animals such as pigs, cattle, and sheep, caused by the foot-and-mouth disease virus (FMDV). Infected animals are primarily characterized by vesicular rashes on the oral mucosa, hooves, and udder skin. The disease spreads rapidly through multiple routes, and each outbreak causes significant economic losses to the local livestock industry. The World Organisation for Animal Health (WOAH) lists it as a nominable infectious disease, and my country also classifies it as the top-ranked animal infectious disease in its category. There are seven main serotypes of FMDV worldwide: O, A, Asian serotypes 1 and 1, and South African serotypes 1, 2, and 3. Vaccination does not provide cross-protection between serotypes. Currently, my country primarily uses vaccination against FMDV serotypes O and A.

[0003] Foot-and-mouth disease (FMD) serotype-specific antibody detection is one of the main serological survey methods used for vaccine efficacy evaluation and epidemiological investigation. Liquid-phase blocking ELISA is a reference method designated by the World Organisation for Animal Health (OIE). Traditional liquid-phase blocking ELISA requires pre-incubation of the test serum and antigen on a carrier reaction plate before transferring the plate to an ELISA plate coated with capture antibodies. Inhibition ELISA, a new method developed by researchers at the Lanzhou Veterinary Research Institute based on the principles of virus neutralization assays, reacts serum antigens directly within the capture antibody ELISA plate, eliminating the need for pre-incubation and shortening the operation time. Double-antibody sandwich ELISA is the FMDV typing method recommended by the World Organisation for Animal Health (WOAH), with serotype-specific monoclonal antibodies as the key reagent. Antibody competition ELISA, developed based on monoclonal antibodies, is simple to operate, time-saving, and has high specificity. Antibody detection in FMD vaccine-immunized animals is a mandatory component of my country's annual FMD prevention and control program.

[0004] Currently, the liquid-phase blocking ELISA method is the foot-and-mouth disease (FMD) immune antibody detection method designated by the Ministry of Agriculture. The inhibition ELISA method, as an upgraded version of the traditional liquid-phase blocking ELISA method, has been widely adopted by veterinary diagnostic laboratories at all levels across the country. Compared with the FMD virus antibody monoclonal antibody competitive ELISA method, the inhibition ELISA method still has user demand for further simplification of operation steps and shortening of operation time. The monoclonal antibody-based FMD virus double antibody sandwich ELISA typing method has the advantages of good sensitivity and high type specificity. The monoclonal antibody competitive ELISA method is not as sensitive as the liquid-phase blocking ELISA method for detecting FMD antibodies, but due to its simple operation steps and short operation time, it is increasingly being used for the detection of FMD antibodies.

[0005] Foot-and-mouth disease virus (FMDV) comprises intact viral particles (146S), an empty capsid (76S), a virus infection-related peptide (45S), and a small protein (12S). The 146S antigen is the main immune component in FMDV vaccines that induces protective antibodies in animals. Polyclonal antibodies prepared by immunizing experimental animals with purified 146S antigen, and monoclonal antibodies prepared using hybridoma screening techniques with mouse myeloma fusion mouse spleen cells, are commonly used as reagents and are employed in liquid-phase blocking ELISA, inhibition ELISA, sandwich ELISA, and monoclonal antibody competitive ELISA. Monoclonal antibodies are antibodies secreted by cell lines that specifically respond to the VP1 gene protein, the main immunogen of FMDV, through hybridoma screening.

[0006] Optimizing and innovating existing methods is a key issue that needs to be addressed in establishing new methods for foot-and-mouth disease antibody detection. The new method should possess the same sensitivity, specificity, and stability as liquid-phase blocking ELISA and inhibition ELISA, the same ease of operation as monoclonal antibody competing ELISA, and minimize experimental failures caused by operational factors. Summary of the Invention

[0007] The technical problem this invention aims to solve is to provide a novel method for detecting foot-and-mouth disease (FMD) antibodies—a sandwich blocking ELISA method. The basic principle involves serially diluting the test serum and positive / negative control sera in a specific ratio on an ELISA plate coated with FMD-specific antibodies. Immediately afterward, enzyme-labeled antibodies are added, followed by the addition of FMD inactivated virus antigen diluted to the working concentration. At least four wells are set up as virus antigen controls (with an equal volume of serum diluent pre-added to each well). The final reaction volume is 200 μL / well. After incubation and washing, TMB substrate is added. After colorimetric termination, the absorbance is read at OD450 nm. The antibodies in the test serum or positive / negative control sera block the virus antigen from participating in the sandwich reaction. The critical value for 50% blocking of the virus antigen is defined as 50% of the average value of the four virus antigen control wells. The result determination is essentially the same as that of the liquid-phase blocking ELISA method. This invention names this ELISA detection technique the sandwich blocking ELISA method, which can also be called a one-step liquid-phase blocking ELISA method.

[0008] Similarities between liquid-phase blocking ELISA and inhibition ELISA for measuring serum antibodies: The key reagents used in both methods (rabbit anti-foot-and-mouth disease virus serum and viral antigen) are the same.

[0009] The differences between liquid-phase blocking ELISA and inhibition ELISA for measuring serum antibodies: Liquid-phase blocking ELISA requires pre-treating the antigen and antibody on a reaction plate before transferring them to the ELISA plate, and the operation time is 2 days; Inhibition ELISA requires diluting the test serum directly on the ELISA plate before adding the viral antigen, and the entire operation of inhibition ELISA can be completed within 2 hours; Sandwich blocking ELISA completes the reaction of the test serum, positive and negative control serum, enzyme-labeled antibody, and viral antigen in one step, taking 30 minutes.

[0010] This invention is the first in China and abroad to break through the application of liquid phase blocking ELISA, inhibition ELISA, monoclonal antibody competitive ELISA and double antibody sandwich ELISA methods. For the first time, it simplifies the complex operation steps of the liquid phase blocking ELISA method into one step, and the detection sensitivity is consistent with that of liquid phase blocking ELISA. It improves the detection specificity and is a more ideal and applicable ELISA method for foot-and-mouth disease antibody detection.

[0011] To achieve this objective, the technical solution adopted by the present invention includes the following steps:

[0012] 1. Foot-and-mouth disease virus antigen was obtained by demulsifying the inactivated foot-and-mouth disease vaccine using a demulsification method. This antigen was used to prepare antiserum and as a virus antigen for detection.

[0013] 2. Purification of foot-and-mouth disease virus 146S antigen (sucrose density gradient centrifugation);

[0014] 3. Preparation of rabbit anti-foot-and-mouth disease virus serum, guinea pig anti-foot-and-mouth disease virus serum, and positive and negative reference sera;

[0015] 4. Immunization of Balb / c mice, preparation of hybridoma cells, screening of monoclonal antibody cell lines, and preparation of ascites fluid;

[0016] 5. Determination of antibody titer in positive control serum (liquid phase blocking ELISA method);

[0017] 6. Preparation, assay, purification of IgG antibody and HRP labeling of monoclonal antibody ascites fluid;

[0018] 7. Optimized operating procedures;

[0019] 8. Result determination;

[0020] 9. Sensitivity and specificity analysis;

[0021] 10. Compatibility with liquid-phase blocking ELISA methods (sensitivity, specificity).

[0022] Specifically, this invention provides a sandwich blocking ELISA method for detecting foot-and-mouth disease virus antibodies. The method involves diluting the test serum and positive / negative control sera pre-coated with foot-and-mouth disease type-specific antibodies onto an ELISA plate, adding enzyme-labeled antibodies, and setting four wells per plate for a control without serum (the control wells are pre-filled with an equal volume of serum diluent). Finally, viral antigen diluted to the working concentration is added, resulting in a final reaction volume of 200 μL / well. The foot-and-mouth disease type-specific antibodies in the test serum and positive / negative sera block the viral antigen from participating in the sandwich reaction. The critical value for 50% blocking of the viral antigen is represented by 50% of the average OD value of the four wells without serum controls. If this critical value falls between the OD values ​​of two adjacent dilutions of a test serum sample, the antibody titer of that serum sample is taken as the median of those two dilutions, consistent with the determination method of the liquid phase blocking ELISA method.

[0023] In the method described in this invention, preferably, the foot-and-mouth disease virus antigen is a serotype-specific foot-and-mouth disease virus antigen, which can be obtained by inactivating foot-and-mouth disease virus or by demulsifying an inactivated foot-and-mouth disease vaccine. Preferably, the enzyme-labeled antibody can be a mixture of one or more monoclonal antibodies labeled with HRP, or it can be a polyclonal antibody prepared by immunization with 146S antigen and then labeled with HRP.

[0024] Preferably, the method described in this invention specifically includes the following steps:

[0025] (1) Foot-and-mouth disease inactivated vaccine was demulsified to obtain foot-and-mouth disease-specific virus antigen;

[0026] (2) Purification of foot-and-mouth disease virus 146S antigen;

[0027] (3) Preparation of rabbit anti-foot-mouth disease virus serum, guinea pig anti-foot-mouth disease virus serum and positive and negative reference serum;

[0028] (4) Purification of guinea pig anti-foot-and-mouth disease virus serum IgG, preparation of type-specific monoclonal antibodies and HPR labeling;

[0029] (5) Determination of antibody titer in standard positive serum (liquid phase blocking ELISA);

[0030] (6) Determination of the concentrations of rabbit antiserum, enzyme-labeled antibody and viral antigen;

[0031] (7) Optimization of operating procedures;

[0032] (8) Result determination.

[0033] a. ELISA-coated plate

[0034] Dilute the serum type-specific rabbit anti-FMDV serum with 0.05M carbonate buffer (pH 9.6) and coat ELISA plates (100 µl / well). Incubate overnight at room temperature. Wash the plates 5 times with PBST. After drying, vacuum pack and store at 4°C.

[0035] b. Serum dilution

[0036] Add serum diluent to each well of an ELISA plate coated with rabbit anti-foot-and-mouth disease virus serum at a volume of 50 µl. Perform serial 2-fold dilutions of both positive and negative control sera and the test serum within the ELISA plate as required. Each reaction plate should include at least four wells with no serum control (pre-filled with 50 µl of serum diluent). The total volume within each ELISA plate should be 50 µl / well.

[0037] c. Add enzyme-labeled antibody

[0038] After diluting the serum sample, enzyme-labeled antibody was added at 50 μL / well, bringing the reaction volume to 100 μL / well.

[0039] d. Add viral antigen

[0040] Finally, add 100 µl of viral antigen diluted to the working concentration per well, for a final reaction volume of 200 µl / well. Seal the plate, shake, and incubate at 37°C for 30 minutes.

[0041] e. Substrate and Termination Reaction

[0042] Wash the plate 3-5 times with 1x PBST, add TMB substrate solution (50µl / well for single component or 50µl / well for two-component AB solution), incubate at 37°C for 10 minutes, then add 50µl of 2MH2SO4 to each well to stop the reaction. Read the absorbance at 450nm using a microplate reader.

[0043] F. Result Determination

[0044] The threshold value for 50% of the average OD value of the four wells of the viral antigen control was used as the cutoff value for 50% blocking of the viral antigen. The OD values ​​of the corresponding wells of the positive and negative control sera and the tested sera were compared with the cutoff value. The antibody titer of a serum sample was the average of the antibody titers of the wells with OD values ​​slightly higher than the cutoff value and those slightly lower than the cutoff value (two adjacent wells). An antibody titer ≥64 was considered positive for foot-and-mouth disease antibodies.

[0045] In the method described in this invention, the antibody titer of the standard positive serum is determined by a liquid-phase blocking ELISA kit produced by the Lanzhou Veterinary Research Institute.

[0046] In any of the methods described above in this invention, the FMDV antigen includes type O FMDV antigen and type A FMDV antigen.

[0047] This invention utilizes the method of directly diluting positive and negative sera and test sera on an ELISA plate coated with capture antibodies, followed by the addition of an equal volume of enzyme-labeled antibody, and finally adding an equal volume of viral antigen diluted to the working concentration. At least four wells are set up as viral antigen controls (with an equal volume of serum diluent added beforehand). The foot-and-mouth disease (FMD) specific antibodies in the test sera and positive and negative sera block the participation of viral antigen in the double-antibody sandwich ELISA reaction between the enzyme-labeled monoclonal antibody and the capture antibody on the ELISA plate. The determination is based on the liquid-phase blocking ELISA method, with 50% of the average OD value of the viral antigen control wells used as the critical value for 50% blocking of the viral antigen. This successfully establishes a blocking sandwich ELISA method, also known as a one-step liquid-phase blocking ELISA method, which can be used for qualitative and quantitative detection of FMD antibodies.

[0048] Beneficial Effects: This invention creatively solves the problems of cumbersome steps in traditional liquid-phase blocking ELISA methods, addresses the challenge of simultaneous interaction between the test serum, capture antibody, and enzyme-labeled antibody with viral antigens, clarifies the method for obtaining the detection viral antigen from the demulsification of inactivated foot-and-mouth disease vaccine, and eliminates the problems of the need for on-the-spot preparation of various reagents, cumbersome operation, instability, and time consumption during the experimental process. Therefore, this invention successfully establishes a sandwich blocking ELISA method, which is simpler, more time-saving, and more stable than traditional liquid-phase blocking ELISA methods for detecting foot-and-mouth disease antibodies—a one-step liquid-phase blocking ELISA method—providing new technical support for foot-and-mouth disease immune antibody monitoring, vaccine immunization efficacy evaluation, epidemiological surveys, and immunization guidance. Detailed Implementation

[0049] This invention comprehensively analyzes three methods for detecting foot-and-mouth disease (FMD) serum antibodies: liquid-phase blocking ELISA, monoclonal antibody competitive ELISA, and double-antibody sandwich ELISA. The liquid-phase blocking ELISA method requires the test serum and antigen to undergo a sufficient neutralization reaction in a liquid phase before being transferred to an ELISA plate coated with rabbit anti-FMD virus serum as the capture antibody. The inhibition ELISA method involves directly diluting the serum on the ELISA plate, immediately adding the working concentration of antigen, followed by incubation with the detection antibody and the enzyme-labeled antibody. The monoclonal antibody competitive ELISA method involves diluting the serum, adding the enzyme-labeled competitive antibody, and incubating for 30 minutes to develop color. Inspired by this, the author first coated rabbit anti-foot-and-mouth disease virus serum onto an ELISA plate. Then, the test serum and positive / negative control sera were serially diluted (50 μL / well) directly onto the ELISA plate as required. Next, enzyme-labeled detection antibodies (one or more monoclonal antibodies, 50 μL / well) were added. Finally, foot-and-mouth disease virus antigen diluted to the working concentration (100 μL / well) was added, resulting in a final reaction volume of 200 μL / well. The plate was incubated at 37°C for 30 minutes, followed by TMB substrate development for 10 minutes. After stopping the reaction, the absorbance was read at OD450 nm. In the ELISA reaction system, the enzyme-labeled monoclonal antibody and the polyclonal antibody coated on the ELISA plate form a double-antibody sandwich method to bind to the antigen in the reaction system. The foot-and-mouth disease antibodies in the sample serum and positive / negative control sera block the antigen in the reaction system from participating in the sandwich method. The antibody level in the sample is directly proportional to the antigen blocking rate. Based on this principle, this invention has successfully established a new technology for detecting foot-and-mouth disease antibodies. This invention names this ELISA method the sandwich blocking ELISA method for detecting foot-and-mouth disease virus antibodies, which can also be called the one-step liquid-phase blocking ELISA method for detecting foot-and-mouth disease virus antibodies.

[0050] As a sandwich-blocking ELISA method for foot-and-mouth disease antibody detection, it inherits all the advantages of liquid-phase blocking ELISA and inhibition ELISA methods for determining serum antibodies, including qualitative and quantitative analysis, stability, sensitivity, specificity, and batch detection capabilities. It represents a further innovation based on traditional liquid-phase blocking ELISA and inhibition ELISA methods, greatly simplifying the operation steps and shortening the reaction time. As simple and time-saving as monoclonal antibody competitive ELISA methods, the entire operation can be completed within one hour. All key reagents used in the experiment, except for the antigen, are provided in working solution form, reducing the steps of manual preparation by operators and greatly minimizing human error during the experimental process. Using this technology, the author has initially established a sandwich-blocking ELISA method for detecting foot-and-mouth disease type O and type A antibodies.

[0051] Therefore, the foot-and-mouth disease virus antibody sandwich blocking ELISA method established in this invention provides new technical support for foot-and-mouth disease vaccine immune antibody monitoring, vaccine immunization efficacy evaluation, epidemiological surveys, and immunization guidance.

[0052] The present invention will be further illustrated below through experiments and embodiments. It should be understood that these embodiments are for illustrative purposes only and do not limit the scope of protection of the present invention. Those skilled in the art will understand that many changes, modifications, and even equivalent alterations can be made to the present invention within the spirit and scope defined by the claims, and all such changes will fall within the scope of protection of the present invention.

[0053] Example 1: Establishment and application of a sandwich-blocking ELISA method for detecting type O foot-and-mouth disease antibodies.

[0054] 1. Materials and Methods

[0055] 1.1 Preparation of Foot-and-Mouth Disease Virus Antigen and Antiserum

[0056] 1.1.1 FMDV antigen preparation and 146S antigen purification: Foot-and-mouth disease inactivated vaccine (O / Mya98 strain) was mixed with demulsifier in a certain proportion, and after vortexing and separation, it was centrifuged at 3000 rpm and 4℃ for 10 minutes. The lower layer of antigen solution was carefully aspirated. 100 ml of antigen solution was taken and centrifuged at 45000 rpm and 4℃ for 1 h. The precipitate on the bottom wall of the centrifuge tube was resuspended in PBS at pH 7.6 to a volume of 2 ml. The mixture was centrifuged at 35000 rpm and 4℃ for 2.5 h using a sucrose density gradient (45%, 35%, 25%, 15%). 0.5 ml samples were taken and the same peak was combined. The samples were then detected by a 260 nm UV spectrophotometer. The absorption peak (second peak) containing the 146S antigen of intact foot-and-mouth disease virus particles was collected and combined.

[0057] 1.1.2 Type O rabbit anti-foot-and-mouth disease virus serum: Select healthy rabbits weighing about 2.0 kg, emulsify the purified 146S antigen with an equal amount of adjuvant and immunize the rabbits. On the 30th day, blood is excised from the carotid artery and the serum is separated, aliquoted, labeled and frozen at -70℃.

[0058] 1.1.3 Type O guinea pig anti-foot-and-mouth disease virus serum: Guinea pigs were immunized with 146S antigen purified from FMDV, which is homologous to rabbit antiserum. Blood was collected from the heart on day 30 and serum was separated, aliquoted, labeled and stored at -70℃.

[0059] 1.1.4 Preparation of Type O mouse monoclonal antibody

[0060] 1.1.4.1 Balb / c mouse immunization: Eight-week-old Balb / c mice were immunized with 146S antigen purified from FMDV, which is homologous to rabbit antiserum, at a dose of 5 μg / mouse, with an interval of 15 days, for a total of 4 immunizations. Three days before fusion with hybridoma cells SP20, the mice were given an intraperitoneal shock with the antigen.

[0061] 1.1.4.2 Fusion: The day before, feeder layers were prepared using mouse peritoneal lavage fluid DMEM (containing HAT), 3 plates per mouse. Mouse spleen cells were isolated using antibiotic-free and serum-free DMEM medium. The mouse spleen was washed, ground, centrifuged, and rinsed. The number of viable spleen cells was approximately 1.0 * 10^8. The ratio of spleen cells to SP20 cells was 5:1. After centrifugation of the mixed spleen cells and SP20 cells, the mixture was gently tapped and placed in a 37°C water bath. 1 ml of PEG was added every 1 minute, rotating the centrifuge tube as it was added. After the addition was complete, the centrifuge tube was capped and allowed to stand for 1–2 minutes. DMEM addition was then stopped. 1 ml of DMEM was added at 1 minute, 1 ml at 2 minutes, 3 ml at 3 minutes, 10 ml at 4 minutes, and 10 ml at 5 minutes. Add 25 ml of DMEM over 5 minutes, dropwise along the wall. After adding, cap the centrifuge tube, let it stand for 2 minutes, gently invert twice, and let it stand at 37°C for 7 minutes. After centrifugation, suspend the fused cells in DMEM containing 1% penicillin-dip antibiotics, 20% bovine serum peptide, and HAT medium. Add the cells to a 96-well cell culture plate with a feeder layer, 100 μL / well, and incubate at 37°C in a CO2 incubator.

[0062] 1.1.4.3 Detection: One week after fusion, the indirect ELISA method was used for detection. Positive wells with OD values ​​greater than 1.0 that reacted with foot-and-mouth disease type O whole virus antigen and VP1 structural protein antigen, and negative wells that reacted with foot-and-mouth disease type A whole virus antigen and VP1 structural protein antigen, were selected for subclone screening.

[0063] 1.1.4.4 Subclone Screening: One day before screening, a feeder layer (containing HT medium) was placed in a 96-well cell culture plate. Cells from the positive wells selected for subclone screening were fully suspended and counted, diluted at 0.5-0.8 cells / well, and added to the 96-well cell culture plate with the feeder layer, 100 μL / well. After incubation at 37°C for 5 days, single-clone wells were labeled. When the single-clone cell population reached 70% of the wells, detection was performed using indirect ELISA and blocking ELISA. Positive wells with an OD value greater than 1.0 in the indirect ELISA test, reacting with the foot-and-mouth disease type O whole virus antigen and VP1 structural protein antigen, and negative for the foot-and-mouth disease type A whole virus antigen and its VP1 structural protein antigen, were selected. Blocking ELISA was then performed on these wells. Wells with the same sensitivity as the foot-and-mouth disease type O specific positive reference serum and negative for the foot-and-mouth disease type A specific reference serum were selected for further culture.

[0064] 1.1.4.5 Preparation of monoclonal antibody ascites: The selected target monoclonal hybridoma cell line was expanded and cultured, then frozen in liquid nitrogen and used for ascites preparation. Eight-week-old female Balb / c mice were pre-injected with 0.2 ml of Freund's incomplete adjuvant into the peritoneum. Three days later, hybridoma cells were injected, with a cell count of 500,000 to 2 million cells per mouse. The abdominal condition of the mice was observed about 7 days after inoculation. When the abdomen was distended and the ascites was full, the ascites was aspirated.

[0065] 1.1.5 Enzyme-labeled antibodies: The ascites IgG of monoclonal antibodies and the IgG of guinea pig anti-foot-and-mouth disease serum were purified by HRPb labeling using a modified sodium periodate method; after being aliquoted with 50% glycerol, they were stored at -20°C for later use.

[0066] 1.1.6 Type O Foot-and-Mouth Disease Antibody Positive Serum: Hyperimmune serum prepared from cattle immunized with foot-and-mouth disease inactivated vaccine homologous to rabbit antiserum. A booster immunization was performed 30 days after the first immunization, and blood was collected and serum separated 2 weeks after the second immunization. The serum was then aliquoted, labeled, and stored at -70℃.

[0067] 1.1.7 Foot-and-mouth disease antibody-negative serum: This refers to serum from newborn calves that are negative for foot-and-mouth disease antibodies.

[0068] 1.2 Methods

[0069] 2.1 Determination of serum titer of type O foot-and-mouth disease antibody positive serum

[0070] The test was performed using a liquid-phase blocking ELISA kit for foot-and-mouth disease type O antibodies (Lanzhou Veterinary Research Institute).

[0071] 2.2 Determination of working concentration of reagent

[0072] Rabbit anti-foot-and-mouth disease virus serum and enzyme-labeled antibodies were used, and the working concentration of each reagent was determined sequentially with the positive serum antibody titer as a reference.

[0073] 2.3 Determination of working antigen concentration

[0074] Set up a positive serum control for type O and a hyperimmune serum control for type A. The antibody titer of the positive control should be consistent with the known values. The antibody titer of the hyperimmune serum for type A should not exceed 32. Select an appropriate dilution titer of the viral antigen.

[0075] 2.4 Operational Step Optimization

[0076] 2.4.1 Serum dilution and one-step reaction of enzyme-labeled antibody and antigen

[0077] On an ELISA plate coated with rabbit antiserum containing type O foot-and-mouth disease virus, add 50 µl of sample diluent to each well. Serially dilute the negative and positive control sera and the test sera as required by 2-fold. Next, add 50 µl of enzyme-labeled antibody per well. Finally, add 100 µl of type O foot-and-mouth disease virus antigen diluted to the working concentration per well. Each reaction plate should have at least four wells containing virus antigen controls (pre-filled with 50 µl of sample diluent). The final liquid volume in each ELISA plate should be 200 µl per well. Seal the plate with the sealing film, vortex, and incubate at 37°C for 30 minutes.

[0078] 2.4.2 Substrate and Termination Reaction

[0079] Wash the plate 3-5 times with 1x PBST, add 50µl of single-component TMB substrate solution per well, or 50µl of two-component TMB AB solution per well, incubate at 37°C for 10 minutes, then add 50µl of 2MH2SO4 to each well to stop the reaction, and read the absorbance at 450nm wavelength using a microplate reader.

[0080] 2.4.3 Result Judgment

[0081] The cutoff value of 50% of the average OD value of the four wells of the viral antigen control was used as the threshold for 50% blocking of the viral antigen. The OD values ​​of the corresponding wells of the negative and positive control sera and the tested sera were compared with the threshold value. The antibody titer of a serum sample was the average of the antibody titers of the wells with OD values ​​slightly higher than the threshold value and slightly lower than the threshold value (two adjacent wells). An antibody titer ≥64 was considered positive for type O foot-and-mouth disease antibody, <32 was considered negative for type O foot-and-mouth disease antibody, and between 32 and 64 was considered suspicious.

[0082] 2.5 Compatibility with liquid-phase blocking ELISA methods (sensitivity and specificity)

[0083] Serum samples from animals immunized with type O foot-and-mouth disease inactivated vaccine (10 from cattle, 10 from sheep, and 10 from pigs), 30 bovine serum samples from animals immunized with type A foot-and-mouth disease inactivated vaccine, and 30 samples from animals negative for foot-and-mouth disease antibodies (15 from cattle and 15 from pigs) were analyzed in parallel with the liquid-phase blocking ELISA method.

[0084] 2.6 Repeatability

[0085] Serum samples from 30 animals immunized with foot-and-mouth disease type O inactivated vaccine were repeatedly tested using a sandwich blocking ELISA method.

[0086] 3 Results

[0087] 3.1 Results of antibody titer assay for positive serum of type O foot-and-mouth disease

[0088] The antibody titer of O-type foot-and-mouth disease positive serum was determined to be 720 (512~1024) by liquid phase blocking ELISA. As a positive control serum for sandwich blocking ELISA, its antibody titer was also controlled at 720 (512~1024).

[0089] 3.2 Results of Antiserum Working Concentration Determination

[0090] The working concentration of rabbit antiserum type O was 1:1000 and the working concentration of enzyme-labeled antibody was 1:5000, as determined by liquid-phase blocking ELISA. Rabbit antiserum type O was coated onto ELISA plates at the working concentration and stored at 4°C; enzyme-labeled antibody was diluted to the working concentration and stored at 4°C.

[0091] 3.3 Results of Viral Antigen Concentration Measurement

[0092] The OD value of the 4 wells for the viral antigen control should be no less than 1.0, the antibody titer of the positive serum should be 720, and the cross-reactivity with its type A foot-and-mouth disease hyperimmune serum should not exceed 32. If the working concentration of the viral antigen is too high, the antibody titer of the positive serum will decrease, and the sensitivity will decrease; if the working concentration of the viral antigen is too low, the antibody titer of the positive serum will increase, the cross-reactivity with other types of hyperimmune serum will increase, and the specificity will decrease.

[0093] 3.4 Optimization Results of Operation Steps

[0094] To ensure sufficient reaction of antigen, enzyme-labeled antibody, and serum, the reaction time of serum, enzyme-labeled antibody, and antigen on the ELISA plate is 37℃ for 30 minutes; to ensure that the OD value of the antigen control is not less than 1.0, the TMB substrate colorimetric reaction is carried out for 10 minutes.

[0095] 3.5 Consistency results with liquid-phase blocking ELISA method (sensitivity and specificity)

[0096] The Lanzhou Veterinary Research Institute's Foot-and-Mouth Disease (FMD) O-type liquid-phase blocking ELISA antibody detection kit indicates a positive result for FMD O antibodies with an antibody titer ≥128. For the sandwich blocking ELISA method, an antibody titer ≥64 indicates a positive result for FMD O antibodies. Serum antibody titer determination was performed on 30 FMD antibody-negative animals and 30 animals immunized with FMD O-type inactivated vaccine. The difference in antibody titers between the sandwich blocking ELISA method and the liquid-phase blocking ELISA method was within one titer, with a sensitivity concordance rate of 100%. For 30 animals immunized with type A inactivated vaccine (all type A antibody titers >1024), the false positive detection rate was 10% for the liquid-phase blocking ELISA method and 1% for the sandwich blocking ELISA method, indicating that the sandwich blocking ELISA method has higher detection specificity than the liquid-phase blocking ELISA method.

[0097] 3.6 Repeatability

[0098] Thirty samples of inactivated foot-and-mouth disease type O vaccine were tested three times using the inhibition ELISA method. 26 samples had completely consistent antibody titers, 3 samples differed by less than one titer, and 1 sample differed by more than one titer, indicating that the reproducibility of this method is very good.

[0099] 4. Conclusion

[0100] 4.1 The sandwich-blocking ELISA method for determining type O foot-and-mouth disease (FMD) antibodies established in this study has good compatibility with liquid-phase blocking ELISA; while maintaining the same sensitivity, it improves the detection specificity. Using the sandwich-blocking ELISA method, an antibody titer ≥64 is considered positive for type O FMD antibodies, consistent with the immunization qualification criteria stipulated for FMD antibody monitoring.

[0101] 4.2 The sandwich blocking ELISA method for detecting type O foot-and-mouth disease antibodies takes less than 1 hour for the complete operation. Compared with the liquid phase blocking ELISA method, which requires overnight reaction of antigen and antibody, and even if it does not take overnight, it still takes more than 5 hours, which greatly improves the detection speed.

[0102] 4.3 Sandwich Blocking ELISA Method for Detecting Type O Foot-and-Mouth Disease Antibodies: During kit development, rabbit anti-foot-and-mouth disease virus serum was pre-coated onto the ELISA plate, and the enzyme-labeled antibody was provided as a working solution. The test serum, enzyme-labeled antibody, and viral antigen are reacted in a single step within a single reaction system, reducing user errors and experimental failures caused by cumbersome reagent preparation and procedures. This kit simplifies the operation of traditional liquid-phase blocking ELISA kits to the extreme.

[0103] 4.4 Obtaining the detection antigen from the demulsification of the inactivated foot-and-mouth disease vaccine expands the source of foot-and-mouth disease virus antigen and ensures its safety. A small amount of foot-and-mouth disease virus antigen is purified by continuous ultracentrifugation followed by sucrose density gradient centrifugation, reducing antigen loss caused by pre-concentration and solving the problem of purifying and preparing antiserum with limited antigen quantities.

[0104] 4.5 This study provides a rapid, sensitive, specific, and stable new detection technology for determining type O foot-and-mouth disease immune antibodies.

[0105] Example 2: Establishment and application of a sandwich-blocking ELISA method for detecting type A foot-and-mouth disease antibodies.

[0106] 1. Materials and Methods

[0107] 1.1 Preparation of Foot-and-Mouth Disease Virus Antigen and Antiserum

[0108] 1.1.1 FMDV antigen preparation and 146S antigen purification: Foot-and-mouth disease inactivated vaccine (AF72 strain) was mixed with demulsifier in a certain proportion, and after vortexing and separation, it was centrifuged at 3000 rpm and 4℃ for 10 minutes. The lower layer of antigen solution was carefully aspirated. 100 ml of antigen solution was taken and centrifuged at 45000 rpm and 4℃ for 1 h. The precipitate on the bottom wall of the centrifuge tube was resuspended in PBS at pH 7.6 to a volume of 4 ml. The solution was centrifuged at 35000 rpm for 2.5 h using a sucrose density gradient (45%, 35%, 25%, 15%). 0.5 ml samples were taken and the same peak was combined. The samples were then detected by a 260 nm UV spectrophotometer. The absorption peak (second peak) containing the 146S antigen of intact foot-and-mouth disease virus particles was collected and combined.

[0109] 1.1.2 Type A rabbit anti-foot-and-mouth disease virus serum: Select healthy rabbits weighing about 2.0 kg, emulsify the purified 146S antigen with an equal amount of adjuvant and immunize the rabbits. On the 30th day, blood is excised from the carotid artery and the serum is separated, aliquoted, labeled and frozen at -70℃.

[0110] 1.1.3 Type A guinea pig anti-foot-and-mouth disease virus serum: Guinea pigs were immunized with 146S antigen purified from FMDV, which is homologous to rabbit antiserum. Blood was collected from the heart on day 30 and serum was separated, aliquoted, labeled and stored at -70℃.

[0111] 1.1.4 Preparation of Type A mouse monoclonal antibody

[0112] 1.1.4.1 Balb / c mouse immunization: Eight-week-old Balb / c mice were immunized with 146S antigen purified from FMDV, which is homologous to rabbit antiserum, at a dose of 5 μg / mouse, with an interval of 15 days, for a total of 4 immunizations. Three days before fusion with hybridoma cells SP20, the mice were given an intraperitoneal shock with the antigen.

[0113] 1.1.4.2 Fusion: The day before, feeder layers were prepared using mouse peritoneal lavage fluid DMEM (containing HAT), 3 plates per mouse. Mouse spleen cells were isolated using antibiotic-free and serum-free DMEM medium. The mouse spleen was washed, ground, centrifuged, and rinsed. The number of viable spleen cells was approximately 1.0 * 10^8. The ratio of spleen cells to SP20 cells was 5:1. After centrifugation of the mixed spleen cells and SP20 cells, the mixture was gently tapped and placed in a 37°C water bath. 1 ml of PEG was added every 1 minute, rotating the centrifuge tube as it was added. After the addition was complete, the centrifuge tube was capped and allowed to stand for 1–2 minutes. DMEM addition was then stopped. 1 ml of DMEM was added at 1 minute, 1 ml at 2 minutes, 3 ml at 3 minutes, 10 ml at 4 minutes, and 10 ml at 5 minutes. Add 25 ml of DMEM over 5 minutes, dropwise along the wall. After adding, cap the centrifuge tube, let it stand for 2 minutes, gently invert twice, and let it stand at 37°C for 7 minutes. After centrifugation, suspend the fused cells in DMEM containing 1% penicillin-dip antibiotics, 20% bovine serum peptide, and HAT medium. Add the cells to a 96-well cell culture plate with a feeder layer, 100 μL / well, and incubate at 37°C in a CO2 incubator.

[0114] 1.1.4.3 Detection: One week after fusion, the indirect ELISA method was used for detection. Positive wells with OD values ​​greater than 1.0 that reacted with foot-and-mouth disease type A whole virus antigen and VP1 structural protein antigen, and negative wells that reacted with foot-and-mouth disease type O whole virus antigen and its VP1 structural protein antigen, were selected for subclone screening.

[0115] 1.1.4.4 Subclone Screening: One day before screening, feeder layers (containing HT medium) were placed in 96-well cell culture plates. Cells from the positive wells selected for subclone screening were fully suspended and counted, diluted at 0.5-0.8 cells / well, and added to the feeder-coated 96-well cell culture plates at 100 μL / well. After 5 days of static incubation at 37°C, single-clone wells were labeled. When the single-clone cell population reached 70% of the wells, detection was performed using indirect ELISA and blocking ELISA. Positive wells with an OD value greater than 1.0 in the indirect ELISA test, reacting with Foot-and-Mouth Disease Type A whole virus antigen and VP1 structural protein antigen, and negative for Foot-and-Mouth Disease Type O whole virus antigen and VP1 structural protein antigen, were selected. Blocking ELISA was then performed on these wells. Wells with the same sensitivity as the Foot-and-Mouth Disease Type A specific positive reference serum and negative for the Foot-and-Mouth Disease Type O specific reference serum were selected for further culture.

[0116] 1.1.4.5 Preparation of monoclonal antibody ascites: The selected target monoclonal hybridoma cell line was expanded and cultured, then frozen in liquid nitrogen and used for ascites preparation. Eight-week-old female Balb / c mice were pre-injected with 0.2 ml of Freund's incomplete adjuvant into the peritoneum. Three days later, hybridoma cells were injected, with a cell count of 500,000 to 2 million cells per mouse. The abdominal condition of the mice was observed about 7 days after inoculation. When the abdomen was distended and the ascites was full, the ascites was aspirated.

[0117] 1.1.5 Enzyme-labeled antibodies: The ascites IgG of monoclonal antibodies and the IgG of guinea pig anti-foot-and-mouth disease serum were purified by HRPb labeling using a modified sodium periodate method; after being aliquoted with 50% glycerol, they were stored at -20°C for later use.

[0118] 1.1.6 Type A Foot-and-Mouth Disease Antibody Positive Serum: Hyperimmune serum prepared from cattle immunized with foot-and-mouth disease inactivated vaccine homologous to rabbit antiserum. A booster immunization was performed 30 days after the first immunization, and blood was collected and serum separated 2 weeks after the second immunization. The serum was then aliquoted, labeled, and stored at -70℃.

[0119] 1.1.7 Foot-and-mouth disease antibody-negative serum: This refers to serum from newborn calves that are negative for foot-and-mouth disease antibodies.

[0120] 2 methods

[0121] 2.1 Determination of serum titer of type A foot-and-mouth disease antibody

[0122] The assay was performed using a liquid-phase blocking ELISA kit for foot-and-mouth disease type A antibodies (Lanzhou Veterinary Research Institute).

[0123] 2.2 Determination of the working concentration of antiserum

[0124] Rabbit anti-foot-and-mouth disease virus serum, guinea pig anti-foot-and-mouth disease virus serum, and enzyme-labeled antibodies were used to determine the working concentration of each reagent in sequence, with the positive serum antibody titer as a reference.

[0125] 2.3 Determination of working antigen concentration

[0126] Set up a positive serum control for type A, and hyperimmune serum controls for type O and Asian type 1. The antibody titer of the positive control should be consistent with the known values. The antibody titers of the hyperimmune serum for type O and Asian type 1 should not exceed 64. Select an appropriate dilution titer of the viral antigen.

[0127] 2.4 Operational Step Optimization

[0128] 2.4.1 Serum dilution and one-step reaction of enzyme-labeled antibody and antigen

[0129] On an ELISA plate coated with rabbit antiserum for foot-and-mouth disease virus type A, add 50 µl of sample diluent to each well. Serially dilute the negative and positive control sera and the test sera as required by 2-fold. Next, add 50 µl of enzyme-labeled antibody per well. Finally, add 100 µl of foot-and-mouth disease virus type A antigen diluted to the working concentration per well. Each reaction plate should have at least four wells with virus antigen controls (pre-filled with 50 µl of sample diluent). The final liquid volume in each ELISA plate should be 200 µl per well. Seal the plate with sealing film, shake, and incubate at 37°C for 30 minutes.

[0130] 2.4.2 Substrate and Termination Reaction

[0131] Wash the plate 3-5 times with 1x PBST, add 50µl of single-component TMB substrate solution per well, or 50µl of two-component TMB AB solution per well, incubate at 37°C for 10 minutes, then add 50µl of 2MH2SO4 to each well to stop the reaction, and read the absorbance at 450nm wavelength using a microplate reader.

[0132] 2.4.3 Result Judgment

[0133] The threshold value for 50% inhibition of the viral antigen was defined as 50% of the average OD value of the four wells in the viral antigen control group. The OD values ​​of the corresponding wells in the negative and positive control sera and the tested sera were compared with this threshold value. The antibody titer of a serum sample was calculated as the average of the antibody titers in wells with OD values ​​slightly above and slightly below the threshold value (between two adjacent wells). An antibody titer >90 was considered positive for type A foot-and-mouth disease antibody, ≤32 was considered negative, and between 32 and 64 was considered suspicious.

[0134] 2.5 Compatibility with liquid-phase blocking ELISA methods (sensitivity and specificity)

[0135] Serum samples from animals immunized with type A foot-and-mouth disease inactivated vaccine (10 from cattle, 10 from sheep, and 10 from pigs), 30 bovine serum samples immunized with Asiatic type 1 foot-and-mouth disease inactivated vaccine, 30 bovine serum samples immunized with type O foot-and-mouth disease inactivated vaccine, and 30 samples from foot-and-mouth disease antibody-negative animals (15 from cattle and 15 from pigs) were analyzed in parallel with the liquid-phase blocking ELISA method.

[0136] 2.6 Repeatability

[0137] Serum samples from 30 animals immunized with foot-and-mouth disease type A inactivated vaccine were repeatedly tested using an inhibition ELISA method.

[0138] 3 Results

[0139] 3.1 Results of antibody titer assay in positive serum for foot-and-mouth disease type A

[0140] The antibody titer of positive serum for type A foot-and-mouth disease was determined to be 720 (512~1024) by liquid phase blocking ELISA. As a positive control serum for sandwich blocking ELISA, its antibody titer was also controlled at 720 (512~1024).

[0141] 3.2 Results of Antiserum Working Concentration Determination: The working concentration of rabbit antiserum type A was determined by liquid-phase blocking ELISA method. The working concentration of enzyme-labeled antibody was 1:1000. Rabbit antiserum type A was coated with ELISA plates at the working concentration and stored at 4℃; enzyme-labeled antibody was diluted to the working concentration and stored at 4℃.

[0142] 3.3 Results of Viral Antigen Concentration Measurement

[0143] The OD value of the 4 wells for the viral antigen control should be no less than 1.0, the antibody titer of the positive serum should be 720, and the cross-reactivity with its type O foot-and-mouth disease hyperimmune serum should not exceed 32. If the working concentration of the viral antigen is too high, the antibody titer of the positive serum will decrease, and the sensitivity will decrease; if the working concentration of the viral antigen is too low, the antibody titer of the positive serum will increase, the cross-reactivity with other types of hyperimmune serum will increase, and the specificity will decrease.

[0144] 3.4 Optimization Results of Operation Steps

[0145] To ensure sufficient reaction of antigen, enzyme-labeled antibody, and serum, the reaction time of serum, enzyme-labeled antibody, and antigen on the ELISA plate is 37℃ for 30 minutes; to ensure that the OD value of the antigen control is not less than 1.0, the TMB substrate colorimetric reaction is carried out for 10 minutes.

[0146] 3.5 Consistency results with liquid-phase blocking ELISA method (sensitivity and specificity)

[0147] The Lanzhou Veterinary Research Institute's Foot-and-Mouth Disease (FMD) type A liquid-phase blocking ELISA antibody detection kit indicates a positive FMD type A antibody test result with an antibody titer ≥128; the sandwich blocking ELISA kit indicates a positive FMD type A antibody test result with an antibody titer ≥64. Serum antibody titer determination was performed on 30 FMD antibody-negative animals and 30 animals immunized with FMD type A inactivated vaccine. The difference in antibody titers between the sandwich blocking ELISA and liquid-phase blocking ELISA methods was within one titer, with a sensitivity concordance rate of 100%. In 30 animals immunized with type O inactivated vaccine (all type O antibody titers >1024), the false positive detection rate was 10% for the liquid-phase blocking ELISA method and 1% for the sandwich blocking ELISA method, indicating that the sandwich blocking ELISA method has higher detection specificity than the liquid-phase blocking ELISA method.

[0148] 3.6 Repeatability

[0149] Thirty samples of inactivated foot-and-mouth disease type A vaccine were tested three times using the inhibition ELISA method. 26 samples had completely consistent antibody titers, 3 samples differed by less than one titer, and 1 sample differed by more than one titer, indicating that the reproducibility of this method is very good.

[0150] 4. Conclusion

[0151] 4.1 The sandwich-blocking ELISA method for detecting type A foot-and-mouth disease (FMD) antibodies established in this study has good compatibility with liquid-phase blocking ELISA; while maintaining the same sensitivity, it improves the detection specificity. Using the sandwich-blocking ELISA method, an antibody titer ≥64 is considered positive for type A FMD antibodies, consistent with the immunization qualification criteria stipulated for FMD antibody monitoring.

[0152] 4.2 The sandwich blocking ELISA method for detecting type A foot-and-mouth disease antibodies takes less than 1 hour for the complete operation. Compared with the liquid phase blocking ELISA method, which requires overnight reaction of antigen and antibody, and even if it does not take overnight, it still takes more than 5 hours, which greatly improves the detection speed.

[0153] 4.3 Sandwich Blocking ELISA Method for Detecting Foot-and-Mouth Disease Antibodies: In the development of this kit, rabbit anti-foot-and-mouth disease virus serum was pre-coated onto the ELISA plate, and the enzyme-labeled antibody was provided as a working solution. The test serum, enzyme-labeled antibody, and viral antigen are reacted in a single step within a single reaction system, reducing operational errors and experimental failures caused by cumbersome reagent preparation and procedures. This kit simplifies the operation of traditional liquid-phase blocking ELISA kits to the extreme.

[0154] 4.4 Obtaining the detection antigen from the demulsification of the inactivated foot-and-mouth disease vaccine expands the source of foot-and-mouth disease virus antigen and ensures its safety. A small amount of foot-and-mouth disease virus antigen is purified by continuous ultracentrifugation followed by sucrose density gradient centrifugation, reducing antigen loss caused by pre-concentration and solving the problem of purifying and preparing antiserum with limited antigen quantities.

[0155] 4.5 This study provides a rapid, sensitive, specific, and stable new detection technology for determining type A foot-and-mouth disease immune antibodies.

Claims

1. A method for detecting foot-and-mouth disease virus antibodies using a sandwich blocking ELISA reagent for non-diagnostic purposes, characterized in that: After serially diluting the test serum and positive and negative control serums on an ELISA plate coated with foot-and-mouth disease type-specific antibodies, enzyme-labeled antibodies were added, followed by foot-and-mouth disease virus antigen diluted to the working concentration. At least 4 wells were set up for virus antigen control. After incubation and washing, TMB substrate was added. After color development was stopped, the absorbance was read at OD 450 nm. 50% of the average OD value of the virus antigen control wells was taken as the critical value for 50% blocking of virus antigen. In the sandwich blocking ELISA reaction system, the enzyme-labeled antibody and the polyclonal antibody coated in the ELISA plate form a double antibody sandwich method to bind the antigen in the reaction system. The foot-and-mouth disease antibody in the test serum and positive and negative control serum blocks the antigen in the reaction system and participates in the sandwich method. The antibody level in the sample is directly proportional to the antigen blocking rate. The foot-and-mouth disease type-specific antibody is rabbit anti-foot-and-mouth disease virus serum; Includes the following steps: Step 1: Coat the ELISA plate: The serologically specific rabbit anti-foot-and-mouth disease virus serum was diluted with pH 9.6 and 0.05M carbonate buffer and coated onto ELISA plates at 100 µl / well. The plates were incubated overnight at room temperature, washed 5 times with PBST, dried, vacuum-packed, and stored at 4°C. Step 2, serum dilution: Add 50 µl of serum diluent to each well of the ELISA plate coated with rabbit anti-foot-and-mouth disease virus serum. Perform serial 2-fold dilutions of positive and negative control sera and test sera in the ELISA plate as required. Set up at least 4 wells without serum control in each reaction plate and add 50 µl of serum diluent in advance. The system in the ELISA reaction plate is 50 µl / well. Step 3: Add enzyme-labeled antibody After diluting the serum sample, enzyme-labeled antibody was added at 50 μL / well, making the reaction volume 100 μL / well. Step 4: Add viral antigen: Finally, add the viral antigen diluted to the working concentration, 100 µl per well, for a final reaction volume of 200 µl / well; seal the plate, shake, and incubate at 37°C for 30 minutes; Step 5: Substrate and Termination Reaction Wash the plate 3-5 times with 1x PBST, add TMB substrate solution, 50µl / well for single component or 50µl / well for two-component AB solution, incubate at 37°C for 10 minutes, then add 50µl of 2M H2SO4 to each well to stop the reaction, and read the absorbance at 450nm wavelength using a microplate reader. Step Six: Result Determination The critical value of 50% of the average OD value of the four wells of the viral antigen control was used as the threshold value for 50% blocking of the viral antigen. The OD values ​​of the corresponding wells of the positive and negative control sera and the tested sera were compared with the critical value. The antibody titer of a serum sample was the average of the antibody titers of the wells with OD values ​​slightly higher and slightly lower than the critical value. An antibody titer ≥64 was judged as positive for foot-and-mouth disease antibody.

2. The method according to claim 1, characterized in that: The foot-and-mouth disease virus antigen is a serotype-specific foot-and-mouth disease virus antigen, obtained by inactivation of foot-and-mouth disease virus or by demulsification of inactivated foot-and-mouth disease vaccine.

3. The method according to claim 1, characterized in that: The foot-and-mouth disease virus antigens include type O FMDV antigen and type A FMDV antigen.

4. The method according to claim 1, characterized in that: The enzyme-labeled antibody is either a mixture of one or more monoclonal antibodies labeled with HRP, or a polyclonal antibody prepared by immunization with 146S antigen and then labeled with HRP.