A broad-spectrum neutralizing antibody against foot-and-mouth disease virus, a neutralizing antibody competition elisa detection kit and application

Abstract

The application provides a foot-and-mouth disease virus broad-spectrum neutralizing antibody, a neutralizing antibody competition ELISA detection kit and application, and belongs to the technical field of antibodies. The foot-and-mouth disease virus broad-spectrum neutralizing antibody has an amino acid sequence of a heavy chain variable region as shown in SEQ ID NO:1 and an amino acid sequence of a light chain variable region as shown in SEQ ID NO:2. The application further provides a foot-and-mouth disease virus neutralizing antibody competition ELISA detection kit, which comprises the following components: a detection plate with a foot-and-mouth disease virus 146S antigen, a biotin-labeled foot-and-mouth disease virus broad-spectrum neutralizing antibody and an enzyme-labeled avidin. The detection sensitivity of the kit is 100%, the specificity is more than 99.5%, the negative / positive coincidence rate compared with the gold standard method VNT for detecting neutralizing antibodies is 92.8%, and the kit can more accurately reflect the level of neutralizing antibodies in serum.

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to a broad-spectrum neutralizing antibody against foot-and-mouth disease virus, a neutralizing antibody competitive ELISA detection kit, and its application. Background Technology

[0002] Foot-and-mouth disease (FMD) is a highly contagious and dangerous animal disease caused by the foot-and-mouth disease virus (FMDV). The World Organisation for Animal Health (WOAH) lists it as a notifiable animal disease, and my country classifies it as the top-ranked animal infectious disease in its category. FMDV is a small RNA virus with quasi-species characteristics of RNA viruses. It has seven serotypes: O, A, C, Asia 1, SAT1, SAT2, and SAT3. Within the same serotype, different topotypes can be distinguished based on geographical characteristics, and each topotype can further differentiate into different genetic lineages or branches, resulting in a wide range of variations. Currently, the prevalent FMDV serotypes in my country are mainly type O and type A. Serotype O is currently the most dangerous FMDV serotype. In my country, there are three topological types and four lineages of serotype O FMDV: the Southeast Asian topological type (SEA) O / Mya / 98 lineage, the Central and Southeast Asian topological type (ME-SA) O / PanAsia lineage and O / Ind / 2001 lineage, and the Chinese topological type (Cathay) new swine virus lineage. Because different serotypes of FMDV cannot generate cross-immunity, it is difficult to produce broadly neutralizing antibodies (bnAbs) capable of neutralizing different serotypes of the virus under natural infection conditions.

[0003] Furthermore, foot-and-mouth disease (FMD) control primarily relies on traditional inactivated vaccines, gradually reducing outbreaks through mandatory immunization. After completing the vaccination schedule, it's necessary to test the vaccine's immune level, and vaccine manufacturers also need to evaluate the efficacy of each batch. Neutralizing antibodies are the main protective immune component against FMDV infection. The virus neutralization test (VNT) is the "gold standard" method for detecting serum antibody titers and evaluating vaccine efficacy in vitro. VNT antibody levels are directly related to immune challenge protection, but VNT requires live virus, has poor stability and repeatability, and a long testing cycle, thus limiting its widespread use. Solid-phase competitive ELISA (SPC-ELISA) and liquid-phase blocking ELISA (LPB-ELISA) are also methods recommended by the World Organisation for Animal Health (WOAH) for evaluating FMD vaccine efficacy in vitro. LPB-ELISA and SPC-ELISA use inactivated antigens, do not require high-level biosafety laboratories, and are rapid and easy to perform. However, their correlation with neutralizing antibodies is poor, and false positives are possible. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a broad-spectrum neutralizing antibody against foot-and-mouth disease virus, which has neutralizing activity against the G1 and G2 branches and A22 subtype of type A foot-and-mouth disease virus SEA97 lineage, Asia type I foot-and-mouth disease virus, Cathay topology, PanAsia lineage of ME-SA topology, India 2001 lineage of ME-SA topology, and Mya-98 lineage of SEA topology foot-and-mouth disease virus.

[0005] This invention provides a broad-spectrum neutralizing antibody against foot-and-mouth disease virus, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2.

[0006] This invention provides the use of the broad-spectrum neutralizing antibody against foot-and-mouth disease virus in the preparation of reagents for blocking foot-and-mouth disease virus infection or in the preparation of drugs for the prevention and / or treatment of foot-and-mouth disease.

[0007] This invention provides the application of the aforementioned broad-spectrum neutralizing antibody against foot-and-mouth disease virus in the preparation of kits for detecting neutralizing antibodies against foot-and-mouth disease virus and / or evaluating the immunization efficacy of vaccines.

[0008] Preferably, the foot-and-mouth disease virus includes the following serotypes: type A, type O, and Asia1.

[0009] Preferably, type A foot-and-mouth disease virus includes the ASIA topological type SEA97 lineage and / or the A22 subtype;

[0010] Foot-and-mouth disease virus type O includes Cathay topology, ME-SA topology and SEA topology;

[0011] Asia 1 type foot-and-mouth disease virus includes the ASIA topology.

[0012] This invention provides a foot-and-mouth disease virus neutralizing antibody competitive ELISA detection kit, comprising the following components: a detection plate capturing foot-and-mouth disease virus 146S antigen, a biotin-labeled broad-spectrum neutralizing antibody against the foot-and-mouth disease virus, and an enzyme-labeled avidin.

[0013] Preferably, in the detection plate that captures the foot-and-mouth disease virus 146S antigen, the foot-and-mouth disease virus 146S antigen is coated onto the detection plate using foot-and-mouth disease virus monoclonal antibody E32 as the capture antibody.

[0014] Preferably, the capture concentration of the foot-and-mouth disease virus 146S antigen is 0.1–3 μg / mL;

[0015] The coating concentration of the foot-and-mouth disease virus monoclonal antibody E32 is 0.1–2 μg / mL.

[0016] Preferably, it also includes one or more of the following: washing solution, diluent, colorimetric solution, stop solution, positive control serum and negative control serum.

[0017] This invention provides a broad-spectrum neutralizing antibody against foot-and-mouth disease virus, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2. In this embodiment of the invention, viruses from two branches of the FMDV A-type SEA97 lineage, namely G1 (representing strain FMDV A / WH / CHA / 09) and G2 (representing strain FMDV / GDMM / CHA / 2013), as well as the FMDV / F72 vaccine strain (A22 subtype strain), FMDVsiaI / JS / 2005 (GenBank No. EF149009), FMDV O-type Cathay topology (representing strain O / HN / CHA / 93, GenBank No. AJ131468), ME-SA topology PanAsia lineage (representing strain FMDV O / xizang / CHA / 99, GenBank No. AJ539138), and ME-SA topology India 2001 lineage (representing strain FMDV). Neutralization assays were performed on the SEA topological Mya-98 lineage (representative strain FMDV O / Mya / 98, GenBank No. JN998085), and the results showed that the monoclonal antibody PO18-10 was resistant to IC50 of all the above representative strains. 50 <50 μg / mL. This indicates that the monoclonal antibody PO18-10 has broad-spectrum neutralizing activity.

[0018] This invention provides a foot-and-mouth disease virus (FMDV) neutralizing antibody competitive ELISA detection kit, comprising: a detection plate capturing FMDV 146S antigen, biotin-labeled broad-spectrum neutralizing antibody against FMDV, and enzyme-labeled avidin. Using the detection kit developed in this invention, 220 non-immunized healthy animal sera and 106 immunopositive sera were tested, demonstrating high sensitivity and specificity (sensitivity 100% and specificity over 99.5%). Furthermore, compared with the gold standard method for neutralizing antibody detection, VNT detection, the negative / positive concordance rate was 92.8%, indicating that the detection kit provided by this invention can accurately reflect the level of neutralizing antibodies in serum. Attached Figure Description

[0019] Figure 1To determine the cutoff value, sensitivity, and specificity of FMDV O CELISA using ROC analysis; where A: CELISA antibody titers in serum samples from healthy animals (n=220) and positive animals (n=106); antibody titers below 0.9 log10 are counted as 0; B: curves showing the sensitivity and specificity of FMDV O CELISA as a function of the cutoff value; the dashed line represents the selected cutoff value;

[0020] Figure 2 The results show the correlation and concordance rate analysis of FMDV O CELISA and VNT detection results; where A represents the antibody titers detected by the two methods, CELISA and VNT; and B represents the concordance rate of the detection results of the two methods, CELISA and VNT. Detailed Implementation

[0021] This invention provides a broad-spectrum neutralizing antibody against foot-and-mouth disease virus. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1 (MEFRLNWVVLFALLQGVQGEEKLLVESGGGLVQPGGSL RLSCVGSGFLFSRSEISWVRQAPGKGLEWLAVIYSSGEITYYADSVKGRFT ISRDDSQNTAYLQMNSLRTEDTARYYCARDYTYGTECSDKWDGMDLWG PGVEVVVSSAPKTAPLVYPLAPCGRDTSGPNVA), and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2 (MAWTVLLIGLLAVGSGVDSQTVIQEPAMSVSP GGTVTLTCAFSSGSVTTSNYPSWYQQTPGQPPRQLIYQTNRRPTGVPSRFSGAISGNKATLTITGAQAEDEADYFCGLYKSCTNIFGGGTHLTVLGQPKAAPTVNLFPPSSEELGTNKATLVCLISDFY).

[0022] In this invention, the foot-and-mouth disease virus broad-spectrum neutralizing antibody is preferably a monoclonal antibody PO18-10 prepared by screening using single B-cell antibody technology. The preparation method is described in Example 1 of the patent application number 201911022083.3.

[0023] In this invention, the FMDV type O antigenic epitopes recognized by the monoclonal antibody PO18-10 are antigenic site 1 [composed of the VP1 G-H loop (133-157) and the linear epitopes of the VP1 carboxyl terminus (200-213), with key amino acid sites being positions 144, 148, 154 and 208 of the VP1 protein] and site 5 (formed by the interaction of the VP1 GH loop with other adjacent amino acids on the capsid surface, with the key site being amino acid position 149 of VP1). This ensures that the monoclonal antibody PO18-10 can block further cellular infection by the virus.

[0024] This invention provides the use of the broad-spectrum neutralizing antibody against foot-and-mouth disease virus in the preparation of reagents for blocking foot-and-mouth disease virus infection or in the preparation of drugs for the prevention and / or treatment of foot-and-mouth disease.

[0025] In this invention, the foot-and-mouth disease virus preferably includes the following serotypes: type A, type O, and Asia1. Type A foot-and-mouth disease virus preferably includes the SEA97 lineage and / or the A22 subtype. Type O foot-and-mouth disease virus preferably includes the Cathay topology, the ME-SA topology, and the SEA topology. Asia1 foot-and-mouth disease virus preferably includes the ASIA topology. In this embodiment of the invention, viruses from two branches of the FMDVA type SEA97 lineage, namely G1 (representing strain FMDVA / WH / CHA / 09) and G2 (representing strain FMDV A / GDMM / CHA / 2013), as well as the FMDVA / F72 vaccine strain (A22 subtype strain), FMDV AsiaI / JS / 2005 (GenBank No. EF149009), FMDV type O Cathay topology (representing strain O / HN / CHA / 93, GenBank No. AJ131468), ME-SA topology PanAsia lineage (representing strain FMDV O / xizang / CHA / 99, GenBank No. AJ539138), and ME-SA topology India 2001 lineage (representing strain FMDV O / XJ / CHA / 2017, GenBank). Neutralization experiments were conducted using the SEA topological Mya-98 lineage (No. MF461724) (representative strain FMDV O / Mya / 98, GenBank No. JN998085). The results showed that the monoclonal antibody PO18-10 was effective against all representative strains at IC50. 50 <50μg / mL indicates that all samples have neutralizing activity.

[0026] This invention provides the application of the aforementioned broad-spectrum neutralizing antibody against foot-and-mouth disease virus in the preparation of kits for detecting neutralizing antibodies against foot-and-mouth disease virus and / or evaluating the immunization efficacy of vaccines.

[0027] In this invention, the type of foot-and-mouth disease virus is the same as that in the above-described technical solution, and will not be repeated here.

[0028] This invention provides a foot-and-mouth disease virus neutralizing antibody competitive ELISA detection kit, comprising the following components: a detection plate capturing foot-and-mouth disease virus 146S antigen, a biotin-labeled broad-spectrum neutralizing antibody against the foot-and-mouth disease virus, and an enzyme-labeled avidin.

[0029] In this invention, the foot-and-mouth disease virus 146S antigen in the detection plate is preferably coated with foot-and-mouth disease virus monoclonal antibody E32 as the capturing antibody. The capture concentration of the foot-and-mouth disease virus 146S antigen is preferably 0.1–3 μg / mL, more preferably 1 μg / mL. The coating concentration of the foot-and-mouth disease virus monoclonal antibody E32 is preferably 0.1–2 μg / mL, more preferably 0.5 μg / mL. The foot-and-mouth disease virus monoclonal antibody E32 can capture FMDV type O 146S antigen. The foot-and-mouth disease virus monoclonal antibody E32 is a known broad-spectrum reactive single B-cell antibody against FMDV, and can react with representative circulating strains of FMDV type O, Asia1, and A. The amino acid sequences of the heavy and light chain variable regions are described in patent document 201911022083.3. The preferred method for preparing the detection plate containing foot-and-mouth disease virus 146S antigen is as follows: Foot-and-mouth disease virus monoclonal antibody E32 is diluted to a working concentration with coating buffer and added to the detection wells. The plate is coated overnight at 4°C. After washing, the working concentration of foot-and-mouth disease virus 146S antigen is added to the detection wells, and the plate is captured at room temperature for 2 hours. The plate is then washed, blocked with blocking buffer, and residual night is removed. The plate is then vacuum-packed. The coating buffer is preferably 0.05 mol / L pH 9.6 carbonate buffer. The washing buffer is preferably PBST solution. The washing frequency is preferably 2-3 times. The blocking buffer is preferably PBS solution containing 1% BSA and 5% sucrose. The blocking temperature is preferably 36-38°C, more preferably 37°C. The blocking time is preferably 50-70 min, more preferably 60 min.

[0030] In this invention, the biotin and neutralizing antibody in the biotin-labeled broad-spectrum neutralizing antibody against foot-and-mouth disease virus are covalently linked. The avidin in the enzyme-labeled avidin is preferably streptavidin. In this embodiment of the invention, the enzyme-labeled avidin was purchased from Genscript.

[0031] In this invention, the kit preferably further includes one or more of the following: washing solution, diluent, chromogenic solution, stop solution, positive control serum, and negative control serum. The washing solution is a 25-fold stock solution, containing 2000.0g sodium chloride, 50.0g potassium chloride, 725.0g disodium hydrogen phosphate dodecahydrate, 50.0g potassium dihydrogen phosphate, and 125mL Tween 20 per 10000mL of water, with a pH of 7.4. The diluent is preferably a PBST buffer containing 2% goat serum, 1% bovine serum albumin (purity greater than 98%), and 0.02% thimerosal preservative. The PBST buffer is preferably a 0.01mol / L PBS buffer containing 0.05% Tween 20, with a pH of 7.2–7.4. The chromogenic solution is selected adaptively according to the type of enzyme in the enzyme-labeled avidin, for example, TMB chromogenic solution. The stop solution is preferably a strong acid or strong base solution, such as a 0.3M sulfuric acid solution. The positive control serum is preferably obtained by immunizing healthy animals with a foot-and-mouth disease virus inactivated vaccine, separating the serum 21 days after booster immunization, filtering and sterilizing it, adding 20% ​​glycerol to a final concentration, and mixing the resulting serum with antibody preservation solution at a volume ratio of 1:2 before packaging. The negative control serum is preferably obtained by adding 20% ​​glycerol to the serum of healthy animals, mixing the resulting serum with antibody preservation solution at a volume ratio of 1:2 before packaging.

[0032] The detection method for the foot-and-mouth disease virus neutralizing antibody competitive ELISA detection kit of the present invention is not particularly limited, and is performed using a competitive detection kit well known in the art. Judgment method: The mean value of the detection results of the 146S antigen control wells is used as the cutoff value, representing the control OD that blocks 50% antibody binding. 450nm Value. Detection well OD 450nm A value greater than the threshold indicates a negative well, and a value less than or equal to the threshold indicates a positive well. The highest dilution of the positive well in the tested sample is used as the antibody titer of that serum sample. An antibody titer ≥ 1:64 is considered positive for foot-and-mouth disease virus type O antibody; ≤ 1:16 is considered negative; an antibody titer between 1:16 and 1:64 is considered suspicious and requires retesting. If the retest is still suspicious, the result is considered negative.

[0033] In this embodiment of the invention, the foot-and-mouth disease virus neutralizing antibody competitive ELISA detection kit detected 220 healthy animal serum samples and 106 positive serum samples confirmed to be infected with type O foot-and-mouth disease virus, with a sensitivity of 100% and a specificity of 99.5%. Simultaneously, experiments showed that the detection kit had a 92.8% concordance rate with VNT detection results, accurately detecting the level of neutralizing antibodies in serum.

[0034] The following detailed description, in conjunction with embodiments, illustrates a broad-spectrum neutralizing antibody against foot-and-mouth disease virus, a neutralizing antibody competitive ELISA detection kit, and its applications provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] Preparation and Identification of a Broad-Spectrum Neutralizing Antibody PO18-10 for Foot-and-Mouth Disease Virus

[0037] 1) Preparation method: One healthy 3-month-old Large White pig was immunized with 2 ml of FMDV O / HN / CHA / 93 inactivated vaccine (containing 2.5 μg 146S antigen per ml) for the first and second immunizations, with immunizations every month. A third immunization was administered 157 days after the first immunization, immunizing with 2 ml of bivalent inactivated FMDV O type (O / xizang / CHA / 99, containing 2.5 μg 146S antigen per ml) and A type (A / WH / CHA / 09 and A / AF72, containing 2.5 μg of each of the two 146S antigens per ml). Peripheral blood mononuclear cells were isolated 3–5 days after the third immunization, and then FMDV O / MYA / 98 146S antigen-specific single B cells were sorted using flow cytometry (BDAria II, USA) for screening and preparation of broad-spectrum neutralizing antibodies against FMDV. The primers used for nested RT-PCR amplification of the heavy chain in porcine single B cells were: outer-F: GTTCGGCTGAACTGGGTGGTC (SEQ ID NO: 7), outer-R: GGTCACTGRCTCGGGGAAGTAGC (SEQ ID NO: 8), inner-F: GGTGGAGTSTGGRGGAGGCCTG (SEQ ID NO: 9), and inter-R: CAGGGGG CCAGAGGGTAGACC (SEQ ID NO: 10); the primers used for amplification of the light chain were: outer-F: ATGGCCTGGAYCCCTCTCCTGCTC (SEQ ID NO: 11), outer-R: CCTCCAGG TCACSGTCACG (SEQ ID NO: 12), and inter-F: TCCTGTGAGCTGACTCAGCC (SEQ ID NO: 13), and inter-R: GTCACTTATTAGACACACCAGGGTG (SEQ ID NO: 14). In the primer sequences above, S represents C or G, Y represents C or T, and R represents A or G. The FMDV porcine-derived broad-spectrum neutralizing monoclonal antibody PO18-10 includes a heavy chain variable region HV1 and a light chain variable region LV1. The amino acid sequence of the heavy chain variable region HV1 is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region LV1 is shown in SEQ ID NO: 2. The heavy chain variable region (SEQ ID NO: 1) of the antibody...NO: 3, ATGGAGTTTCGGCTGAACTGGGTGGTCTTGTTTGCTCTCTTACAAGGTGTCCAGGGTGAGGAGAAGCTGCTGGTGGAGTCTGGAGGAGGCCTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTCGGCTCTGGATTCCTCTTCAGTCGTAGTGAAATTAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGCTGGCGGTTATTTATAGTAGTGGCGAGATCACCTACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACGACTCCCAGAACACGGCCTATCTGCAAATGAACAGCCTGAGAACCGAAGACACGGCCCGCTATTACTGTGCAAGGGACTATACGTATGGTACTGAGTGTTCCGATAAGTGGGATGGTATGGATCTCTGGGGCCCAGGCGTTGAGGTCGTCGTGTCCTCAGCCCCCAAGACGGCCCCATTGGTCTACCCTCTGGCCCCCTGCGGCAGGGACACGTCTGGCCCTAACGTGGCC) and the light chain variable region (SEQ IDNO: 4, The coding sequences of ATGGCCTGGACGGTGCTTCTGATCGGGCTCCTCGCTGTCGGCTCAGGGGTGGATTCTCAGACTGTGATCCAGGAGCCGGCGATGTCAGTGTCTCCTGGAGGGACCGTCACACTCACCTGTGCCTTTAGCTCTGGGTCAGTCACTACTAGTAACTACCCCAGCTGGTACCAGCAGACACCAGGCCAACCTCCCCGACAGCTGATCTACCAAACAAACAGACGCCCGACTGGGGTCCCCAGTCGCTTCTCTGGAGCCATCTCTGGGAACAAAGCCACCCTCACCATCACGGGGGCCCAGGCTGAGGACGAGGCCGACTACTTCTGTGGTCTGTATAAAAGTTGTACTAATATTTTCGGCGGTGGGACGCATCTGACCGTCCTCGGTCAGCCCAAGGCCGCTCCCACGGTCAACCTCTTCCCGCCCTCCTCTGAGGAGCTCGGCACCAACAAGGCCACCCTGGTGTGTCTAATAAGTGACTTCTACC) are respectively the same as the porcine IgG2 heavy chain (SEQ ID NO: 5: GCCCCAAAGACTGCCCCTTCCGTGTACCCACTGGCTCCTTGCGGAAGGGATACTAGCGGCCCAAATGTCGCTCTGGGCTGTCTGGCCAGCAGCTACTTCCCAGAGCCAGTGACAATGACATGGAACAGCGGAGCTCTGACATCCGGCGTGCACACTTTTCCAAGCGTGCTGCAACCAAGCGGACTGTACTCTCTGAGCTCCATGGTGACAGTCCCAGCCAGCTCCCTCTCCAGCAAGAGCTACACATGCAACGTGAATCACCCAGCCACTACAACTAAGGTCGACAAGAGGGTGGGCACTAAGACTAAACCTCCATGTCCTATTTGCCCCGGCTGCGAAGTGGCCGGACCAAGCGTGTTTATTTTCCCACCTAAGCCAAAGGACACTCTCATGATCAGCCAGACACCAGAGGTCACTTGCGTCGTCGTCGACGTGTCCAAGGAACATGCCGAGGTCCAGTTTAGCTGGTATGTGGACGGCGTCGAAGTGCATACTGCTGAGACAAGGCCTAAGGAGGAGCAGTTTAACTCCACATATAGGGTCGTCTCCGTCCTCCCAATCCAGCATCAAGATTGGCTGAAGGGCAAAGAGTTCAAGTGCAAGGTGAACAACGTGGATCTGCCAGCCCCTATCACAAGGACAATCTCCAAGGCCATTGGCCAGTCTAGGGAGCCACAAGTGTACACTCTGCCACCACCAGCCGAGGAGCTGTCTAGGAGCAAGGTCACTGTGACATGTCTCGTCATCGGCTTCTACCCACCAGATATTCACGTCGAGTGGAAGTCCAACGGACAACCAGAACCAGAGGGCAATTATAGGACTACACCTCCACAGCAAGATGTGGACGGAACATTCTTTCTGTACAGCAAGCTCGCCGTCGACAAAGCTAGGTGGGACCATGGCGAAACATTCGAGTGTGCCGTGATGCACGAGGCTCTGCACAACCACTACACACAGAAGAGCATCTCCAAAACTCAAGGCAAG) and the constant region sequence of the light chain (SEQ IDNO: 6: CAGCCAAAGGCTGCCCCTACAGTGAATCTCTTCCCTCCTAGCT CCGAAGAACTGGGCACTAACAAGGCCACCCTGGTGTGTCTCATCAGCGACTTCTACCCCGGCGCCGTGACTGTGACTTGGAAGGCTGGCGGAACTACTGTGACACAAGGCGTGGAGACTACTAAGCCTAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTATCTGGCTCTGTCCGCCAGCGATTGGAAGTCCAGCTCCGGATTCACTTGTCAAGTGACACACGAAGGAACTATCGTGGAGAAGACTGTGACTCCTTCCGAGTGTGCC) were ligated and inserted into the pcDNA3.4 eukaryotic expression vector. The heavy and light chain expression plasmids were mixed at a mass ratio of 2:3 and transfected into CHO suspension culture cells to obtain complete IgG2 subtype antibodies. The antibodies were purified by affinity chromatography. The preparation method of the antibody PO18-10 is detailed in the patent application number CN201810929067.1.

[0038] 2) Identification methods

[0039] Virus neutralization assay detects the neutralizing activity of antibodies.

[0040] To determine the neutralizing activity of the porcine FMDV monoclonal antibody PO18-10 screened above, the following strains were tested: FMDV strain SEA97 lineage G1 (representative strain FMDVA / WH / CHA / 09) and G2 (representative strain FMDVA / GDMM / CHA / 2013) viruses, as well as the FMDVA / F72 vaccine strain (A22 subtype), FMDVAsiaI / JS / 2005 (GenBank No. EF149009), FMDV O type Cathay topology (representative strain O / HN / CHA / 93, GenBank No. AJ131468), ME-SA topology PanAsia lineage (representative strain FMDV O / xizang / CHA / 99, GenBank No. AJ539138), and ME-SA topology India 2001 lineage (representative strain FMDV). Neutralization tests were conducted on the SEA topological Mya-98 lineage (representative strain FMDV O / Mya / 98, GenBank No. JN998085) (O / XJ / CHA / 2017, GenBank No. MF461724).

[0041] The specific operating steps are as follows:

[0042] ① In a 96-well cell culture plate, serially dilute the monoclonal antibody PO18-10 with cell maintenance medium at a rate of 50 μL / well, from a 1:4 to a 1:512 dilution. Then, add 50 μL of a solution containing 100 TCID to each well. 50 The FMDV was applied at 37°C for 1 hour. Simultaneously, TCID values ​​of 0.1, 1, 10, and 100 were set. 50 Control wells (without antibody).

[0043] ② Add 50 μL of 5 × 10 to each well 4 Complete culture medium for BHK21 cells was placed in an incubator at 37°C with 5% CO2 for 48 hours.

[0044] ③ Discard the supernatant cell solution, add pre-cooled fixative (methanol:acetone = 1:1), and fix at -20℃ for 20 min.

[0045] ④ Discard the fixative and add 100 μL of crystal violet solution to each well for staining. After 30 min, wash the 96-well plate and observe the antibody concentration (IC50) of cells that have not yet developed pathogenesis. 50 ). Use 50 μg / mL IC. 50 The value is used as the critical value for neutralization, and >50 μg / mL is defined as non-neutralizing activity.

[0046] Virus neutralization assay results showed that the monoclonal antibody PO18-10 was effective against all representative strains at IC50. 50 <50μg / mL indicates neutralizing activity.

[0047] Example 2

[0048] A foot-and-mouth disease virus neutralizing antibody competitive ELISA detection kit, its preparation method and usage method

[0049] 1. Components of the reagent kit

[0050] 1) Preparation method of detection plate for capturing 146S antigen:

[0051] Dilute E32 antibody to 0.5 μg / mL with coating buffer (0.05 mol / L pH 9.6 carbonate buffer), add 100 μL to each well of the ELISA plate, and incubate overnight at 4°C. Wash the ELISA plate three times with PBST and blot dry. Dilute FMDV type O 146S antigen (derived from FMDV O / Mya / 98 strain, NBCI accession number JN998085) to 1 μg / mL with PBS (pH=7.8), add 100 μL to each well of the ELISA plate, capture at room temperature for 2 hours, wash three times with PBST, and blot dry. Block with blocking buffer (PBS solution containing 1% BSA and 5% sucrose) at 37°C for 1 hour. Discard the blocking buffer, blow dry the ELISA plate, vacuum pack, include two desiccant packets, and store sealed at 2–8°C.

[0052] Each kit contains 5 reaction plates containing 146S antigen.

[0053] 2) The preparation method of biotin-labeled neutralizing antibody PO18-10 is as follows:

[0054] A1. First, place the monoclonal antibody PO18-10 in a 100kDa ultrafiltration tube and centrifuge at 4000rpm for 10min. Resuspend the monoclonal antibody in PBS buffer.

[0055] A2. Take 180 μL of ultrapure water and add it to 1 mg of biotin, then dilute to 10 mM; add 2 μL of 10 mM biotin to 1 mL of 1 mg / mL monoclonal antibody PO18-10.

[0056] Place A3 or 4℃ on ice for 2 hours.

[0057] A4. Centrifuge the above-obtained reaction system at 4000 rpm for 10 min using an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and resuspend it in antibody storage buffer.

[0058] A5. Add an equal volume of 100% glycerol to monoclonal antibody PO18-10 and store at -70°C.

[0059] Each kit includes one tube (500 μL) of 100× Biotin-labeled monoclonal antibody PO18-10.

[0060] 3) 100× enzyme-labeled streptavidin (Strep-HRP): 1 tube (700μL), which is horseradish peroxidase (HRP) labeled streptavidin. It is a commercially available reagent (Genscript) and is prepared by diluting the enzyme-labeled streptavidin in antibody preservation solution (Sigma). It has a shelf life of up to 1.5 years.

[0061] 4) Preparation of 25× Washing Solution: For two 60mL bottles, add 2000.0g sodium chloride, 50.0g potassium chloride, 725.0g disodium hydrogen phosphate dodecahydrate, and 50.0g potassium dihydrogen phosphate to 5000mL of ultrapure water, and bring the volume to 10000mL. Adjust the pH to 7.4. Autoclave (10Pa, 30min). After the temperature drops to 10-30℃, add 125mL Tween 20 and mix well. Dispense quantitatively, seal, and label. Store at 10-30℃ for later use.

[0062] 5) Serum diluent: Prepare two 60mL bottles of PBST (0.01mol / L PBS buffer containing 0.05% Tween-20, pH 7.2–7.4) containing 2% goat serum, 1% bovine serum albumin (purity >98%), and 0.02% thimerosal preservative under aseptic conditions. After filtration and sterilization, dispense quantitatively, seal, and label.

[0063] 6) TMB substrate solution: 1 bottle (60mL), the TMB substrate is a commercial product (e.g., single-component TMB substrate from SurModics, USA, catalog number TMBW), with a shelf life of 36 months. Under aseptic conditions, quantitatively dispense into sterile brown reagent bottles and store at 2-8℃ for later use.

[0064] 7) Termination solution: 1 bottle (60 mL), a sulfuric acid solution with a final concentration of 0.3 mol / L. Slowly add 16.32 mL of 98% concentrated sulfuric acid to 900 mL of ultrapure water, stirring thoroughly. Finally, add ultrapure water to bring the volume to 1000 mL. Dispense quantitatively, seal, label, and store at 10–30℃ for later use.

[0065] 8) Positive control serum: 1 tube (300μL), blood was collected from healthy animals immunized with FMDV type O inactivated vaccine 21 days after booster immunization to separate serum, filtered and sterilized with a 0.22μm filter, added with 20% sterile glycerol and an appropriate amount of preservative, and then dispensed and stored below -20℃ for later use.

[0066] During kit assembly, positive control serum was diluted 1:2 with commercial antibody preservation solution (e.g., StabilZyme HRP Conjugate Stabilizer, SZ02-2000 from SurModics, USA) under aseptic conditions, quantitatively aliquoted, sealed, and labeled.

[0067] 9) Negative control serum: 1 tube (300 μL), collected from healthy animals without FMDV antibodies under aseptic conditions. After filtration and sterilization, add 20% sterile glycerol to a final concentration and an appropriate amount of preservative, then aliquot and store at -20°C or below. During kit assembly, under aseptic conditions, dilute the negative control serum 1:2 with the aforementioned commercial antibody preservation solution, then quantitatively aliquot, seal, and label.

[0068] 10) Other items: including 5 U-shaped 96-well serum dilution plates, 5 pipette trays, 10 sealing films and 1 instruction manual.

[0069] 2. Usage and judgment methods of the prepared test kit

[0070] S1 Usage

[0071] Open the outer plastic seal of the reagent kit. All reagents should be brought to 10–30°C before use. Gently mix the serum sample using a vortex mixer. Follow the procedure below:

[0072] S1.1 Remove the sealed Capture 146S antigen reaction plate, tear it open along the cut of the packaging bag, and record the sample position.

[0073] S1.2 Preparation of washing solution

[0074] Dilute the 25× detergent solution to 1× detergent solution using deionized water or distilled water.

[0075] S1.3 Diluted Serum

[0076] Add serum diluent to a U-shaped 96-well serum dilution plate, 90 μL / well in the first row and 60 μL / well in the remaining wells; then add the serum sample to be tested and the negative and positive control serum to the wells in the first row, 30 μL / well, and serially dilute (1:4 to 1:512) to a final volume of 60 μL / well; dilute only 4 wells for the negative control, and add only 4 wells for the 146S antigen control with serum diluent.

[0077] S1.4 with biotin-labeled neutralizing antibody

[0078] Dilute 100× Biotin-labeled neutralizing antibody to the working concentration at a ratio of 1:100 using serum diluent, and add 60 μL / well to the U-shaped 96-well serum dilution plate described in S1.3. Double the serum dilution ratio after adding an equal volume of biotin-labeled neutralizing antibody. Gently vortex or pipette mix when transferring to the reaction plate.

[0079] S1.5 transferred to the reaction plate

[0080] Transfer the mixture from the U-shaped 96-well serum dilution plate described in S1.4 to a 146S antigen reaction plate, 100 μL / well. Seal the plate and incubate at 37°C for 1 h. Carefully remove the sealing film, wash the plate 5 times with washing buffer, 300 μL / well, and pat dry for the last time.

[0081] S1.6 with enzyme-labeled avidin

[0082] Dilute 100× enzyme-labeled avidin to the working concentration using serum diluent at a ratio of 1:100, 100 μL / well, seal the plate, and incubate at 37°C for 15 min. Carefully remove the sealing film, wash the plate 5 times with washing buffer, 300 μL / well, and pat dry after the last wash.

[0083] S1.7 Add substrate solution

[0084] Add 100 μL of TMB substrate solution per well, seal the plate, and incubate at 37°C in the dark for 10–15 min.

[0085] S1.8. After the colorimetric reaction is complete, add stop solution (100 μL / well) and read the OD value on a microplate reader. 450nm value.

[0086] S2, Judgment

[0087] S2.1 Result Validity Determination

[0088] The above test results are valid only if the following criteria are met: 146S antigen control OD 450nm The value should be above 1.0; the positive control serum titer should be ≥1:256; the negative control serum antibody titer should be ≤1:16.

[0089] S2.2 Calculation of antibody titer in serum samples

[0090] 146S antigen control (4 wells), discard the highest and lowest OD values. 450nm Value, calculate the average OD of the remaining 2 wells. 450nm The value, divided by 2, is the critical value, representing the control OD that blocks 50% antibody binding. 450nm Value. Detection well OD 450nm A value greater than the threshold indicates a negative well, and a value less than or equal to the threshold indicates a positive well. The highest dilution of the positive well in the tested sample is used as the antibody titer of that serum sample. An antibody titer ≥ 1:64 is considered positive for foot-and-mouth disease virus type O antibody; ≤ 1:16 is considered negative; an antibody titer between 1:16 and 1:64 is considered suspicious and requires retesting. If the retest is still suspicious, the result is considered negative.

[0091] Example 3

[0092] 1. Serum sample

[0093] Healthy non-immune bovine serum (negative serum): A total of 220 samples were tested and found to be negative for antibodies against O, A, and Asia 1 structural proteins, as well as negative for antibodies against non-structural protein 3ABC.

[0094] Positive serum samples: 106 serum samples were identified as positive for FMDV type O by VNT.

[0095] Immune swine serum: A total of 40 samples were collected from 10 pigs each of the laboratory-prepared inactivated vaccines of FMDV SCGH / CHA / 2016 (Cathay topology, GenBank No. KX161429), FMDV O / xizang / CHA / 99, FMDV O / XJ / CHA / 2017, and FMDV O / Mya / 98 strains. Blood samples were collected on day 21 after the second immunization.

[0096] Immunized bovine serum: A total of 50 samples were collected from 12 or 13 cattle immunized with each of the laboratory-prepared inactivated vaccines of FMDV SCGH / CHA / 2016, FMDV O / xizang / CHA / 99, FMDV O / XJ / CHA / 2017, and FMDV O / Mya / 98 strains. Blood was collected on day 21 after the second immunization.

[0097] Immunization serum from sheep: A total of 50 samples were collected from 12 or 13 cattle immunized with inactivated vaccines of the FMDV SCGH / CHA / 2016, FMDV O / xizang / CHA / 99, FMDV O / XJ / CHA / 2017, and FMDV O / Mya / 98 strains prepared in the laboratory. Blood was collected on the 21st day after the second immunization.

[0098] 2. Determination of optimal reaction conditions for the competitive ELISA detection method using FMDV type O neutralizing antibody

[0099] E32 (0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, 2 μg / mL), biotin-labeled PO18-10 (0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, 2 μg / mL), and 146S antigen (0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, 2 μg / mL) were determined by square matrix titration. The working concentrations of E32, biotin-labeled PO18-10, 146S antigen, and enzyme-labeled avidin (1:5000, 1:10000, 1:20000, 1:30000, 1:40000, 1:50000) were determined to be 0.5 μg / mL, 1 μg / mL, 1 μg / mL, and 1:30000, respectively.

[0100] 3. Operational Procedure for FMDV Type O Neutralizing Antibody Competitive ELISA Detection Method

[0101] ① Coating: Dilute monoclonal antibody E32 to 0.5 μg / mL with coating buffer (0.05 mol / L carbonate buffer, pH 9.6), add 100 μL to each well of the microplate, and incubate overnight at 4°C. Wash the microplate three times with PBST and blot dry.

[0102] ② Antigen capture: FMDV type O 146S antigen was captured with monoclonal antibody E32. The antigen was diluted with PBS to the working concentration (1 μg / mL), and 100 μL was added to each well of the ELISA plate. The plate was reacted at room temperature for 2 h, washed 3 times with PBST, and then patted dry.

[0103] ③ Blocking: Add 100 μL of blocking solution (PBS solution containing 1% BSA and 5% sucrose) to each well, block at 37°C for 1 h, discard the solution, and dry for storage.

[0104] ⑤ Competitive reaction: Positive, negative control sera, and test serum samples were serially diluted (1:4–1:512) into a serum dilution plate using serum dilution buffer (PBST buffer containing 2% goat serum, 1% bovine serum albumin, and 0.02% thimerosal preservative), 60 μL per well. Four wells were left undiluted as 146S antigen controls. 60 μL of biotin-labeled monoclonal antibody PO18-10 diluted to a working concentration (1 μg / mL) with serum dilution buffer was added to each well, and the mixture was gently vortexed. 100 μL of the mixture from each well was transferred to the ELISA plate and incubated at 37°C for 1 h. The plate was then washed five times with 1×PBST washing buffer, and finally patted dry.

[0105] ⑥ Add enzyme-labeled streptavidin: Dilute the enzyme-labeled streptavidin to the working concentration (1:30000) with serum diluent, add 100 μL to each well of the ELISA plate, react at room temperature for 15 min, wash 5 times with PBST, and pat dry.

[0106] ⑦ Add substrate for color development: Add 100 μL of TMB substrate solution to each well and react at room temperature in the dark for 10–15 min.

[0107] ⑧ Termination: Add 0.3 mol / L H2SO4 to each well to terminate the reaction; measure the OD value at 450 nm wavelength using an enzyme-linked immunosorbent assay (ELISA) reader.

[0108] ⑨ Calculate serum antibody titers: 4 wells of 146S antigen control, discarding the highest and lowest OD values. 450nm Value, calculate the average OD of the remaining 2 wells. 450nm The value, divided by 2, is the critical value, representing the control OD that inhibits the response by 50%. 450nm Value. Detection well OD 450nm A value greater than the threshold indicates a negative well, and a value less than the threshold indicates a positive well. The highest dilution factor of the positive wells in the tested sample is used as the antibody titer of that serum sample.

[0109] Determination of critical values ​​and results of specificity and sensitivity measurements

[0110] The method established in this invention was used to detect 220 serum samples from non-immune healthy animals and 106 serum samples from animals whose VNT was determined to be positive for FMDV type O. The detection results are shown in [Figure 1]. Figure 1According to ROC curve analysis, when the cut-off value is 1.5log10, the AUC has the largest area of ​​0.9998 (SE = 0.0001926, 95% confidence interval 0.9995–1.000, P < 0.0001), at which point the sensitivity is 100% and the specificity is 99.5%. Figure 1 (B) Based on the VNT determination results, the determination criteria for this kit are as follows: when the antibody titer of the sample to be tested is ≤1.2log10(1:16), it is determined to be negative serum; when the antibody titer is ≥1.8log10(1:64), it is determined to be positive; when the antibody titer is between 1.2Log10(1:16) and 1.8Log10(1:64), it is determined to be suspicious and requires retesting. If the retest is still suspicious, it is determined to be negative.

[0111] Example 4

[0112] Virus micro-neutralization test (VNT)

[0113] The virus neutralization experiment was performed on serum samples from immunized or infected animals using FMDV O / Mya / 98. The specific experimental steps are as follows:

[0114] ① In a 96-well cell culture plate, serially dilute the serum sample to be tested with cell maintenance medium at a rate of 50 μL / well, from a 1:4 dilution to 5:12. Then, add 50 μL of a solution containing 100 TCID to each well. 50 The FMDV was applied at 37°C for 1 hour. Simultaneously, TCID values ​​of 0.1, 1, 10, and 100 were set. 50 Control wells (without serum sample).

[0115] ② Add 50 μL of 5 × 10 to each well 4 Complete culture medium for BHK21 cells was placed in an incubator at 37°C with 5% CO2 for 48 hours.

[0116] ③ Discard the supernatant cell solution, add pre-cooled fixative (methanol:acetone = 1:1, volume ratio), and fix at -20℃ for 20 min.

[0117] ④ Discard the fixative and add 100 μL of crystal violet solution to each well for staining. After 30 min, wash the 96-well plate and observe the maximum dilution of serum with 50% non-pathogenic cells. The maximum serum dilution is used to represent the virus neutralizing antibody titer.

[0118] The method established in this embodiment was used to test 139 serum samples from pigs, cattle, and sheep immunized with FMDV type O inactivated vaccine. The serum samples were also validated using VNT. The results are shown below. Figure 2A. A comparative analysis of the results from the two methods showed that the negative / positive concordance rate between the CELISA and VNT detection results was 92.8% (129 / 139)%. Figure 2 (B) This indicates that the competitive ELISA kit for detecting neutralizing antibodies established in this invention can accurately reflect the level of neutralizing antibodies in serum.

[0119] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A broad-spectrum neutralizing antibody against foot-and-mouth disease virus, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

2. The use of the broad-spectrum neutralizing antibody against foot-and-mouth disease virus as described in claim 1 in the preparation of a medicament for the prevention and / or treatment of foot-and-mouth disease caused by infection with foot-and-mouth disease virus type A, O, or Asia 1.

3. The use of the broad-spectrum neutralizing antibody against foot-and-mouth disease virus according to claim 1 in the preparation of a kit for detecting neutralizing antibodies against foot-and-mouth disease virus and / or evaluating the immunization effect of foot-and-mouth disease virus vaccine, wherein the foot-and-mouth disease virus is at least one of the following serotypes: type A, type O, and Asia1.

4. The application according to claim 2 or 3, characterized in that, Type A foot-and-mouth disease virus includes the ASIA topological type SEA97 lineage and / or the A22 subtype; Foot-and-mouth disease virus type O includes Cathay topology, ME-SA topology and SEA topology; Asia 1 type foot-and-mouth disease virus includes the ASIA topology.

5. A foot-and-mouth disease virus neutralizing antibody competitive ELISA detection kit, characterized in that, It comprises the following components: a detection plate that captures the 146S antigen of foot-and-mouth disease virus type O serotype, a biotin-labeled broad-spectrum neutralizing antibody for foot-and-mouth disease virus as described in claim 1, and an enzyme-labeled avidin; The foot-and-mouth disease virus is at least one of the following serotypes: type A, type O, and Asia1.

6. The foot-and-mouth disease virus neutralizing antibody competitive ELISA detection kit according to claim 5, characterized in that, The O serotype foot-and-mouth disease virus 146S antigen was coated onto the detection plate using foot-and-mouth disease virus monoclonal antibody E32 as a capture antibody. The monoclonal antibody E32 includes a heavy chain variable region HV1 and a light chain variable region LV1. The amino acid sequence of the heavy chain variable region HV1 is MNPLWTLLFVLSAPRGVLSQVQLRESGPSLVKPSQTLSLTCTVSGFSLSNYALNWVRQAPGKALECLGGIGPSGHTDYNPALKSRLIITKDNSKNQVSLSVSSVTPEDTATYICARDSGIYGTSGWGCIGGFDDNYIDAWGHGLLVTVSS. The amino acid sequence of the light chain variable region LV1 is MSTMAWSPLFLTLVAVYTGSWAQAVLTQPSSVSGSLGQRVSITCSGSSNNIGAHGVGWYQQIPGSGLRTIIYNNSKRPSGVPDRFSGSKSGNTATLTISSLQAGDEADYFCATSDYSTRSSAFGSGTTLTVLGQPKSAPSVTLFPPSKEELSANK.

7. The foot-and-mouth disease virus neutralizing antibody competitive ELISA detection kit according to claim 6, characterized in that, The capture concentration of the O serotype foot-and-mouth disease virus 146S antigen is 0.1~3 μg / mL; The coating concentration of the foot-and-mouth disease virus monoclonal antibody E32 is 0.1~2 μg / mL.

8. The foot-and-mouth disease virus neutralizing antibody competitive ELISA detection kit according to any one of claims 5 to 7, characterized in that, It also includes one or more of the following: washing solution, diluent, colorimetric solution, stop solution, positive control serum, and negative control serum.

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