Hybridoma cell strain secreting type a foot-and-mouth disease monoclonal antibody and its application

Abstract

The application belongs to a hybridoma cell strain secreting A type foot-and-mouth disease monoclonal antibody and application thereof.The monoclonal antibody secreted by the hybridoma cell strain disclosed in the application has high serum type specificity, and the A type foot-and-mouth disease virus cELISA antibody detection method based on the monoclonal antibody can specifically detect A type foot-and-mouth disease virus antibody, avoid cross reaction between virus serum types, and is suitable for susceptible livestock pigs, cattle and sheep, and has high precision.The monoclonal antibody prepared in the application can cover all A type foot-and-mouth disease virus inactivated vaccine antibodies produced in China at present, and has no cross reaction with O type foot-and-mouth disease virus strain standard positive serum and other common susceptible viruses of odd-toed livestock, has wide coverage, and the detection result is higher than that of existing similar monoclonal antibodies and detection methods.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a hybridoma cell line that secretes a monoclonal antibody against foot-and-mouth disease type A and its applications. Technical Background

[0002] Foot-and-mouth disease (FMD) is a major animal disease caused by the foot-and-mouth disease virus (FMDV). It primarily infects cloven-hoofed animals such as pigs, cattle, and sheep, and has high morbidity and mortality rates. The World Organisation for Animal Health (WOAH) classifies it as an animal disease requiring global notification, and my country also classifies it as a Class A animal disease. FMDV belongs to the Picornaviridae family, FMDV genus, and has 7 serotypes (O, A, Asia-1, C, SAT1, SAT2, and SAT3) and more than 70 subtypes. There is no cross-immunoprotection between serotypes.

[0003] The most common method for evaluating the immunization efficacy of foot-and-mouth disease (FMD) vaccines is the ELISA serum antibody detection method, which differentiates serotypes. This method is further divided into competitive methods, liquid-phase blocking methods, and indirect methods. Among them, the competitive method is considered a relatively accurate method and is the international trade diagnostic method designated by the World Organisation for Animal Health (WOAH). Currently, commercially available FMD virus ELISA antibody detection methods mostly use complete inactivated antigens and their prepared polyclonal antibodies, preparing different antibody detection kits for different serotypes. However, due to inherent defects in polyclonal antibody diagnostic reagents, non-specific reactions easily occur between serotypes or the coverage spectrum within serotypes is narrow. This is mainly manifested in the following ways: 1. In the commonly used O and A type FMD virus antibody detection technologies (kits) in China, non-specific cross-reactions between serotypes are prone to occur, leading to inaccurate assessment of FMD virus antibody levels in different serotypes; 2. Single serotype antibody detection technologies cannot cover antibodies produced by all strains within a serotype, especially for commonly used vaccine strains in China, which can easily lead to missed detections and misdiagnosis within the same serotype due to different inoculated strains.

[0004] Monoclonal antibody preparation technology has been developed for decades and has been widely used in the treatment and detection of many viral diseases. Among them, some research has been carried out on foot-and-mouth disease virus antibody detection methods based on monoclonal antibodies. In 2020, Grazioli et al. established a monoclonal antibody-based ELISA serum antibody kit for foot-and-mouth disease virus types O, A, C and Asia 1 (Transboundary and Emerging Diseases, 2020, 67(6):3005-3015). However, the preparation and validation of their monoclonal antibodies were based on the main circulating strains in Europe, and their specificity and sensitivity were not validated by standard positive sera of the main circulating strains in Asia.

[0005] Chinese patent CN110144329B discloses "Hybridoma cell line 6B1 and its secreted monoclonal antibody against foot-and-mouth disease type A virus and its application." The ELISA detection method established by this invention has a low specificity, with a negative detection rate of 90% (195 / 216) for known porcine type A negative samples and known porcine type O antibody positive samples. Furthermore, the sample validation spectrum of this invention is narrow, limited to porcine serum for negative samples, lacking validation data for negative sera from cattle and sheep, which are also susceptible to foot-and-mouth disease. Since there are significant differences in antibody background between bovine and sheep serum and porcine serum, it is difficult to determine its specificity detection effect on bovine and sheep serum. In addition, this invention lacks cross-reactivity data for the currently important domestic vaccine strains OS / 99 (type O cathay genetic lineage) and O / HB / HK / 99 (type O PanAsia genetic lineage). The antigens and antibodies of these two strains are readily available, and due to the high variability of foot-and-mouth disease virus, specificity validation for these two viruses is necessary.

[0006] Chinese patent CN111172118A discloses "Hybridoma cell line of anti-A type foot-and-mouth disease antigen monoclonal antibody and its application". This invention only uses one indirect ELISA for screening, and does not describe in detail the viruses and methods used in the screening process. It only involves O type Myanmar 98, Os99 Pan-Asian lineage I, A type Akto 58, Asia I Jiangsu 05, and Asia I Kizilsu 03 virus antigens in the Western blot verification process. The O and A type strains do not cover the major circulating strains in China, and it has been announced that there is no Asia I type foot-and-mouth disease virus circulating in China. The production and scientific research of related vaccines have been prohibited since 2017. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a hybridoma cell line that secretes a monoclonal antibody against type A foot-and-mouth disease virus and its applications.

[0008] This invention provides a hybridoma cell line secreting a monoclonal antibody against foot-and-mouth disease virus type A, named: Mouse Spleen-Myeloma Cell Hybridoma Cell Line, with accession number CGMCC No. 46034; deposit address: China General Microbiological Culture Collection Center; deposit date: August 8, 2024. Deposit address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The present invention also provides a foot-and-mouth disease type A virus-specific monoclonal antibody A10H2-F2 secreted by the foot-and-mouth disease type A virus hybridoma cell line CGMCCNo.46034.

[0010] This invention also provides the application of the foot-and-mouth disease type A virus Mc10H2-F2 hybridoma cell line in the detection of foot-and-mouth disease virus. The detection includes antigen detection or antibody detection. The detection can specifically detect foot-and-mouth disease type A virus antigens and antibodies, and is not reactive to foot-and-mouth disease type O virus and other viral antigens and antibodies.

[0011] This invention also provides the application of the foot-and-mouth disease type A virus-specific monoclonal antibody A10H2-F2 in the detection of foot-and-mouth disease virus. The detection includes antigen detection or antibody detection. The detection can specifically detect foot-and-mouth disease type A virus antigens and antibodies, and is not reactive to foot-and-mouth disease type O virus and other viral antigens and antibodies.

[0012] Furthermore, the foot-and-mouth disease type A virus includes strains AKT-Ⅲ, AF / 72, and Re-A / WH / 2009; the foot-and-mouth disease type O virus includes strains Mya98 / xJ / 2010, OHM / 02, HB / HK / 99, GX / 09-7, and OS / 99; and the other viruses include classical swine fever virus, porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus, porcine circovirus, pseudorabies virus, bluetongue virus, bovine viral diarrhea virus, poxvirus, and akaban virus.

[0013] This invention also provides a method for preparing a hybridoma cell line that secretes monoclonal antibodies against foot-and-mouth disease virus type A, comprising the following steps:

[0014] Prokaryotic expression and purification of S1 and A type foot-and-mouth disease virus VP1 proteins;

[0015] S2. The purified VP1 protein obtained in S1 was used as an antigen to immunize mice. After immunization, the spleen of the mice was removed and fused with myeloma cells to obtain aggregated hybridoma cells.

[0016] S3. Initial screening: When the clustered hybridoma cell clones described in S2 grow and occupy more than 20% of the bottom area of ​​the well, the initial screening is performed using an ELISA method with Re-A / WH / 2009 strain inactivated virus as the coating antigen. OD450 ≥ 0.4 is judged as a positive clone, and the positive clones are transferred to a 24-well culture plate for further culture.

[0017] S4. Second screening: When the positive clone hybridoma cells cultured in S3 grow to a density of 50% to 80%, a second screening is performed. The supernatant of each well is used to inactivate the virus with Re-A / WH / 2009 strain and to detect the antigen coated with BHK-21 cells containing 10% serum in an indirect ELISA plate. Hybridoma cell lines that can react with a mixture of serum and BHK-21 cells are eliminated, and hybridoma cell lines that can only react with foot-and-mouth disease virus are expanded to a larger scale.

[0018] S5. Third Screening: When the hybridoma cells expanded from S4 grow to a density of 50%–80%, a third screening is performed, namely competitive ELISA screening. Standard positive sera of type A AKT-Ⅲ, AF72, Re-A / WH / 2009 strains and type O Mya98 / xJ / 2010, OHM / 02, HB / HK / 99, GX / 09-7, OS / 99 strains and hybridoma cell supernatant are used to competitively react with the coated A / WH / 2009 inactivated virus. Hybridoma cell lines whose supernatant is competitive with antibodies against all the above type A strains (i.e., inhibition rate ≥60%), and has no or low competitiveness with all type O strains (i.e., inhibition rate ≤40%) are selected. These hybridoma cell lines secrete monoclonal antibodies against type A foot-and-mouth disease virus.

[0019] The beneficial effects of this invention are as follows:

[0020] 1. This invention discloses a hybridoma cell line that secretes a monoclonal antibody specific to type A foot-and-mouth disease virus. The secreted monoclonal antibody has high serotype specificity. The cELISA antibody detection method for type A foot-and-mouth disease virus established based on this monoclonal antibody can specifically detect type A foot-and-mouth disease virus antibody, avoid cross-reaction between viral serotypes, and is applicable to foot-and-mouth disease susceptible livestock such as pigs, cattle, and sheep, thus significantly improving the accuracy of detection technology.

[0021] 2. The monoclonal antibody prepared by this invention can cover all inactivated vaccine antibodies against type A foot-and-mouth disease virus currently produced in China, including strains such as AKT-Ⅲ, AF72, and Re-A / WH / 2009. It shows no cross-reaction with standard positive sera of type O foot-and-mouth disease virus strains Mya98 / xJ / 2010, OHM / 02, HB / HK / 99, GX / 09-7, and OS / 99, as well as other common susceptible viruses in cloven-hoofed livestock.

[0022] 3. The hybridoma cell line provided by this invention grows vigorously and is easy to culture and cryopreserve. Monoclonal antibodies can be obtained naturally through hybridoma cell culture or by inoculating hybridoma cells into the peritoneal cavity of mice and then aspirating peritoneal fluid. Monoclonal antibody preparation is relatively easy.

[0023] 4. The monoclonal antibody prepared based on the hybridoma cell line of the present invention and the cELISA (competitive enzyme-linked immunosorbent assay) antibody detection method for type A foot-and-mouth disease virus established on this basis can specifically detect antibodies against the main circulating strains and vaccine strains of type A foot-and-mouth disease virus in China, covering the main susceptible livestock of foot-and-mouth disease, such as pigs, cattle and sheep. This can improve the accuracy of vaccine immunization effect evaluation and distinguish between antibodies produced by wild virus infection and vaccine immunization.

[0024] 5. This invention uses the expressed VP1 structural protein of type A foot-and-mouth disease virus to prepare monoclonal antibodies. The VP1 structural protein is the main serotype-specific protein of the virus, while existing technologies use whole virus particles, making this invention more targeted. Furthermore, in the screening process for type A foot-and-mouth disease virus serotype-specific monoclonal antibodies, this invention uses a three-step screening method. Different ELISA methods (indirect ELISA and competitive ELISA) are used to gradually eliminate non-specific and weakly specific monoclonal antibodies. In the third screening process, standard positive sera of foot-and-mouth disease virus are used for inhibition rate verification and screening. The standard positive sera cover the main O and A strains currently used in domestic vaccines, making them suitable for ELISA antibody detection methods and for evaluating vaccine immunization efficacy. The monoclonal antibodies prepared and the established ELISA antibody detection method of this invention are applicable to the detection of type A foot-and-mouth disease virus antibodies in pigs, cattle, and sheep, with broad coverage. Compared with the results of the neutralization test, a classic evaluation method for foot-and-mouth disease virus antibodies, the positive and negative serum concordance rate is 97.3%, higher than existing monoclonal antibodies and detection methods of the same type. Attached Figure Description

[0025] Figure 1 Electrophoresis image for verification of pSumo-mut-AVP1 vector;

[0026] Figure 2 SDS-PAGE electrophoresis image of the VP1 protein expression product identification results;

[0027] Figure 3 Western blot analysis of VP1 protein expression product identification results;

[0028] Figure 4 The results are the initial screening ELISA test results for Example 3;

[0029] Figure 5 This is a schematic diagram of a specific monoclonal antibody screening procedure;

[0030] Figure 6 The results of the typing and identification of A10H2-F2 monoclonal antibodies;

[0031] Figure 7 Distribution of inhibition rates in known type A foot-and-mouth disease virus antibody-negative sera;

[0032] Figure 8 The results are from the competitive ELISA detection of standard positive serum for foot-and-mouth disease in Example 6. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] The foot-and-mouth disease virus strains involved in this invention, AKT-Ⅲ, AF72, Re-A / WH / 2009, Mya98 / xJ / 2010, OHM / 02, HB / HK / 99, GX / 09-7, and OS / 99, were all legally procured or donated by Zhongpu Biopharmaceutical Co., Ltd. and Jinyu Baoling Biopharmaceutical Co., Ltd. Standard positive sera were prepared by the National Foot-and-Mouth Disease Professional Laboratory (Kunming).

[0035] The methods described in this invention should be understood as not being limited to the specific methods and experimental conditions described herein, as these methods and conditions can be changed. Furthermore, the terminology used herein is only for describing specific implementation schemes and not for limitation; some terms are explained below:

[0036] Monoclonal antibody: The "monoclonal antibody" mentioned in this invention refers to an antibody secreted by hybridoma cells that targets a single antigenic epitope of a virus, which is understood by those skilled in the art.

[0037] Specificity: The "specificity" mentioned in this invention refers to the ability to specifically identify a single virus or a single serotype of the same virus, which is understandable to those skilled in the art.

[0038] Susceptible animals: refers to cloven-hoofed animals that can be infected with foot-and-mouth disease virus, mainly including pigs, cattle, sheep, etc.

[0039] Inoculation: refers to the injection of an inactivated vaccine into the subcutaneous, intramuscular, or peritoneal cavity of an animal using a syringe, in a manner that can be understood by a person generally skilled in the art.

[0040] OD450: refers to the absorbance at a wavelength of 450nm when using an ELISA reader or other similar instruments or equipment.

[0041] The descriptions of foot-and-mouth disease virus names, ELISA, cELISA, specificity tests, and sensitivity tests in this invention are all general methods that can be understood by those skilled in the art.

[0042] Example 1: Prokaryotic expression and purification of type A foot-and-mouth disease virus VP1 protein:

[0043] 1.1 Selection and amplification of the target expression fragment:

[0044] The complete VP1 gene sequence A / HuBWH / CHA / 2009(JF792355) was selected and downloaded from the NCBI website. The complete VP1 gene fragment (636bp) was synthesized by Qingke Biotechnology Co., Ltd. VP1-specific primers with added protective bases and restriction enzyme sites were designed and synthesized. The synthesized gene fragment was amplified using the PrimeScript High Fidelity RT-PCR kit from Takara Bio. The amplified product was purified using a DNA purification kit and stored at -80℃ for later use.

[0045] 1.2 Construction of the pSumo-mut-AVP1 expression vector:

[0046] The pSumo-mut plasmid carrying the 6HIS tag was purchased from Zhongding Biotechnology Co., Ltd. HindIII and XhoI restriction exonucleases were used to simultaneously digest the pSumo-mut plasmid and the target gene fragment amplified in section 1.1. After electrophoresis, the target fragment was recovered by gel extraction, yielding two linear DNA fragments with lengths of approximately 5600 bp and 656 bp, respectively. The two linear DNA fragments were mixed appropriately and ligated overnight at room temperature using T4 ligase. The ligation products were transformed into DH5α competent cells and plated on nutrient agar plates containing IPTG, X-Gal, and Kanmycin. The plates were incubated upside down at 37°C. Single white bacterial strains were selected for liquid nutrient agar expansion, and PCR detection and electrophoresis verification were performed using pSumo-mut plasmid primers. Figure 1 As shown, successful amplification of the fragment and correct size (750bp) indicates successful ligation, thus obtaining the pSumo-mut-AVP1 expression vector intended for expressing the VP1 protein of type A foot-and-mouth disease virus.

[0047] 1.3 Expression and validation of VP1 protein of foot-and-mouth disease virus type A:

[0048] The constructed pSumo-mut-AVP1 expression vector plasmid was transformed into BL21(DE3) competent cells (purchased from Tiangen Biotech Co., Ltd.) for kanamycin resistance selective culture. The selected bacterial strain was added at 1% to liquid LB medium containing 100M kanamycin and incubated at 37°C with shaking for 3–4 hours until the OD600 value was between 0.6 and 0.8. Then, 0.2mM IPTG was added to a final concentration and the culture was incubated overnight at 15°C. The cultured bacterial solution was centrifuged at 5000g for 15 minutes at 4°C, the supernatant was discarded, and the precipitate was resuspended in 50mM Tris-HCl buffer (1.0% Triton X-100, pH 8.0). The bacterial cells were disrupted on ice using an ultrasonic disruptor, and then centrifuged at 12000g for 30 minutes at 4°C. The liquid was collected by filtration through a 0.45nm filter, purified using a nickel column, and analyzed by SDS-PAG and Western blot.

[0049] The results of the identification of VP1 protein expression products are shown in the figure. Figure 2 and Figure 3 In the SDS-PAGE electrophoresis diagram, M represents the protein molecular weight standard, 1 represents 0.5 mg / mL BSA, and 2 represents the purified sample. In the Western blot analysis diagram, M represents the protein molecular weight standard, and 1 represents the purified sample. Figure 2 and Figure 3 The results showed bands at approximately 43 kDa, with the fragments larger than the expected 37.48 kDa. Analysis indicated this was due to changes in protein structure, which would not affect the subsequent preparation and screening of monoclonal antibodies. The purified protein was aliquoted and stored at -80°C for later use.

[0050] Example 2: Immunization and hybridoma cell preparation in Balb / C mice:

[0051] 2.1 Immunization of mice:

[0052] Six Balb / C mice aged 6-8 weeks were selected. The purified VP1 protein from Example 1 was emulsified 1:1 with Freund's adjuvant and administered subcutaneously at multiple sites at weeks 0, 2, 5, and 8, with 0.8 mL per mouse per injection. The initial immunization used complete Freund's adjuvant, while subsequent immunizations used incomplete Freund's adjuvant. At week 11, tail amputation was performed, and serum was collected for antibody testing. A titer greater than or equal to 128 was considered acceptable. Mice that passed the initial test underwent a pulse immunization at week 13, with 0.8 mL of purified antigen injected intraperitoneally per mouse. The immunization schedule is shown in Table 1. Mice were purchased from the Experimental Animal Center of Kunming Medical University, and the experiment was conducted at the Experimental Animal Center of Yunnan Biopharmaceutical Co., Ltd.

[0053] Table 1 Immunization schedule for mice

[0054]

[0055] 2.2 Hybridoma cell preparation:

[0056] Five days after intraperitoneal injection of the antigen, mice were euthanized by cervical dislocation, and the spleen was removed. The spleen cells were minced and fused into RPMI 1640 medium. Hybridoma cells were prepared by fusing them with myeloma cells (SP2 / 0 cells) according to the method described in the "Laboratory Guide to Antibody Preparation and Use" (GC Howard and MR Kaiser, translated and published by Science Press). The fused cell suspension was added to 96-well cell culture plates, 100 μL per well, and incubated at 37°C with 5% CO2. After 20 hours, 100 μL of RPMI 1640 complete medium containing twice the amount of HAT was added to each well. Subsequently, 50% of the RPMI 1640 complete medium containing 1 part HAT was replaced every 3 days for a total of 3 replacements. Afterward, a complete medium replacement was performed using RPMI 1640 complete medium without HAT. The selective culture procedure for hybridoma cells is shown in Table 2.

[0057] Table 2 Selective Culture Procedures for Hybridoma Cells

[0058]

[0059]

[0060] Example 3: Screening for specific monoclonal antibodies:

[0061] The specific monoclonal antibody screening procedure of this invention is as follows: Figure 5 As shown, it includes the following steps:

[0062] 3.1 Initial Screening:

[0063] After the selective culture procedure for hybridoma cells in Example 2 is completed, when agglomerated, adherent hybridoma cell clones appear in most wells, occupying more than 30% of the bottom area of ​​each well, the first screening is performed using an indirect ELISA method. The specific method is as follows:

[0064] (1) The purified Re-A / WH / CHA / 2009 strain inactivated virus (a gift from Jinyu Baoling Biopharmaceutical Co., Ltd.) was diluted to approximately 2 μg / mL with 0.02M PBS (pH 7.6), and added to a 96-well ELISA plate at a rate of 50 μL / well. The plate was placed in a sealed humidified chamber and incubated at 4–6°C for 18–24 h. The liquid was then poured off and the plate was patted dry. 300 μL of washing buffer was added to each well, and the plate was incubated for 1 min before being discarded and patted dry. This washing process was repeated twice to complete the antigen coating. The washing buffer consisted of 0.01M PBS with 0.05% TWEEN-20 added. The purified and inactivated Re-A / HuBWH / CHA / 2009 strain virus was purchased from Zhongpu Biopharmaceutical Co., Ltd.

[0065] (2) Under sterile conditions, use a pipette to draw 50 μL of hybridoma cell supernatant and transfer it to the above-mentioned ELISA plate. The cell culture wells correspond one-to-one with the wells of the ELISA plate. Incubate at 37°C for 60 minutes, then discard the liquid inside and wash 3 times as above.

[0066] (3) Dilute the goat HRP-labeled anti-mouse secondary antibody 1:1000, 50 μL per well, incubate at 37°C for 30 minutes, and wash 5 times as above. The HRP-labeled goat anti-mouse secondary antibody was purchased from Beijing Solarbio Biotechnology Co., Ltd.

[0067] (4) Add 50 μL of single-component TMB chromogenic solution to each well, incubate at 37°C in the dark for 10 minutes, then add 1.25% sulfuric acid solution to terminate the reaction. Read the OD value (absorbance) at 450 nm wavelength using a microplate reader within 10 minutes. See the results below. Figure 4 A yellow color reaction indicates a positive antibody test, while a negative test shows no color. The single-component TMB chromogenic solution was purchased from Surmodics, product number TMBWX-1000-01.

[0068] (5) Cell clusters with OD values ​​≥ 0.4 and clearly formed by the expansion of single-cell clones were selected for amplification culture. A total of 80 positive clones were screened and transferred to 24-well culture plates for further culture. The culture medium was RPMI 1640 complete medium containing 10% fetal bovine serum.

[0069] 3.2 Second screening:

[0070] When the positive clone hybridoma cells from step 3.1 reached a density of 50%–80% adherent in 24-well culture plates, a second screening (specific screening) was performed. ELISA plates were coated with Re-A / WH / CHA / 2009 virus-inactivated antigen and BHK-21 cells containing 10% serum, respectively. The monoclonal antibody to be tested was used for indirect ELISA detection with the two different coating antigens, following the same procedure as described in step 3.1. Hybridoma cell lines that showed a positive reaction with the serum and BHK-21 cell mixture were discarded. The hybridoma cell lines that showed a positive reaction only with foot-and-mouth disease virus were expanded to a larger scale. The second screening identified 26 hybridoma cell lines that showed a positive reaction to the Re-A / WH / CHA / 2009 foot-and-mouth disease virus antigen and showed no cross-reactivity with bovine serum and BHK-21. These were then transferred to 6-well plates for further culture in RPMI 1640 complete medium containing 10% fetal bovine serum after resuscitation.

[0071] 3.3 Third screening:

[0072] The hybridoma cells cultured in section 3.2 above were subjected to a third screening (serotype-specific competitive ELISA, cELISA) when they adhered to the wells of a 24-well culture plate and grew to a density of 50%–80%. Nine standard sera, including AKT-III strain, AF72, Re-A / WH / CHA / 2009 strain, and O-type Mya98 / xJ / 2010, OHM / 02, HB / HK / 99, GX / 09-7, and OS / 99 strains (bovine-derived positive and negative sera), were used to compete with antibodies secreted by the 26 hybridoma cell lines in the ELISA reaction. The standard sera were prepared by the National Foot-and-Mouth Disease Laboratory (Kunming). Specific operating steps:

[0073] (1) The coating method for the Re-A / WH / CHA / 2009 strain in the 96-well ELISA plate is the same as described in 3.1 above;

[0074] (2) Add standard serum to the ELISA plate at a volume of 10 μL / well. Make 26 replicates for each standard serum. Add 50 μL / well of the supernatant of 26 hybridoma cell culture medium to the wells containing standard serum. That is, each hybridoma cell culture medium is mixed with 9 kinds of standard serum and competitively reacts with the coated antigen. Incubate at 37°C for 60 minutes, then discard the liquid and wash 3 times as above.

[0075] (3) Subsequent operating steps are the same as in 3.1;

[0076] (4) The inhibition rate (PI) of the standard positive serum against the monoclonal antibody was calculated according to the formula: Inhibition rate (PI) = (1 - serum OD value / negative serum OD value) × 100%. Hybridoma cell lines that showed competitive activity with antibodies against all A-type virus strains (inhibition rate ≥ 60%) and showed no or low competitive activity with antibodies against all O-type virus strains (inhibition rate ≤ 40%) were selected. These hybridoma cell lines secreted monoclonal antibodies against type A foot-and-mouth disease virus were obtained. A comprehensive analysis of the growth status of the hybridoma cells, the amount of monoclonal antibody secreted, and the specific response effect were conducted to finally obtain a vigorous growth hybridoma cell line of type A foot-and-mouth disease virus Mc10H2-F2 that could stably secrete type A specific monoclonal antibodies. The inhibition rate is shown in Table 3.

[0077] Table 3. Inhibition rate of standard serum against foot-and-mouth disease type A virus Mc10H2-F2 hybridoma cell line

[0078] Re-A / WH / 2009 Type A 77.95％ A / AF / 72 Type A 73.12％ A / AKT-III Type A 86.66％ Mya98 / xJ / 2010 O type 26.87％ OHM / 02 O type 35.47％ HB / HK / 99 O type 26.56％ GX / 09-7 O type 31.94％ OS / 99 O type 29.36％ Standard negative serum / 0.00％ .

[0079] Example 4: Identification of Monoclonal Antibody Subtypes:

[0080] Roche IsoStrip application TMThe mouse monoclonal antibody typing kit was used to identify the subtypes of monoclonal antibodies. The procedure was followed according to the kit instructions. Results are shown below. Figure 6 The A10H2-F2 monoclonal antibody was identified as the IgG1 subtype, with the light chain being the κ subtype.

[0081] Example 5: Establishment and determination of the cutoff value for cELISA detection of foot-and-mouth disease virus type A based on monoclonal antibodies:

[0082] The purified Re-A / WH / 2009 strain (20 μg / mL) was diluted 1:40 with coating buffer (pH 7.6) using 0.02 M PBS (pH 7.6), and then added to a 96-well ELISA plate at a rate of 50 μL / well. The plate was incubated at 4–6 °C for 20 h, the supernatant was discarded, and the plate was washed twice with PBST (0.01 M PBS + 0.05% Tween 20, pH 7.2–7.4). After draining, blocking buffer was added and the plate was blocked at room temperature for 4 h. The blocking buffer was discarded, and the plate was air-dried, sealed, and stored at 4 °C for later use. Based on a fixed antigen coating amount, the antibody diluent components, the amount of sample added, the dilution factor of the single batch of A10H2-F2 monoclonal antibody, the dilution factor of the single batch of goat anti-mouse HRP-labeled secondary antibody, and the TMB reaction time were determined through matrix cross-validation experiments. The optimized results are shown in Table 4.

[0083] Table 4. Reaction conditions for cELISA antibody detection of Foot-and-Mouth Disease Virus Type A

[0084]

[0085] Using optimized reaction conditions and reagent ratios, serum samples from 120 known negative pigs, cattle, and sheep were tested. The inhibition rate was calculated using the formula: inhibition rate = 1 - (sample to be tested / negative control) × 100%. The cutoff value for result determination was then established. The detection and analysis results are shown below. Figure 7 The results showed that the average inhibition rate was... The percentage was 22.8%, and the standard deviation (SD) was 8.4%. The number of known negative samples with an inhibition rate ≥40% is 0. Therefore, the threshold value is set at 40%. That is, an inhibition rate ≥40% is considered positive for type A foot-and-mouth disease virus (FMDV) antibody, otherwise it is negative.

[0086] Example 6: Specificity test of cELISA antibody detection method:

[0087] Table 5 shows the results of standard antibody positive serum tests for monoclonal antibody A10H2-F2 against common ungulate viruses CSFV, PRRSV, PPV, PCV, PRV, BTV, BVDV, PoxV, and AKAV. The results indicate that monoclonal antibody A10H2-F2 did not exhibit significant competitive reaction with the antibodies against the aforementioned viruses, and all were deemed negative based on a 40% cutoff value.

[0088] Table 5. Validation results of cELISA detection method for the specificity of multiple viral antibodies.

[0089]

[0090] Note: 40% is used as the cutoff value for positive and negative results. PC is the positive control and PN is the negative control. "-" indicates negative FMDV antibody of type A and "+" indicates positive FMDV antibody of type A.

[0091] The results of the detection of the monoclonal antibody A10H2-F2 with standard positive serological samples of type O and type A foot-and-mouth disease virus are shown in the figure. Figure 8 Based on the fact that the monoclonal antibody A10H2-F2 secreted by this hybridoma cell line can inhibit the reaction between antibodies of type A foot-and-mouth disease (FMDV) vaccine strains AKT-Ⅲ, AF / 72, and Re-A / WH / 2009 and the coating antigen, while it cannot inhibit the reaction between antibodies of type O foot-and-mouth disease (FMDV) vaccine strains Mya98, PanAsia, and CATHAY and the coating antigen, this detection method can specifically detect antibodies of common and prevalent type A foot-and-mouth disease virus strains in my country, and there is no cross-reaction with antibodies of common and prevalent type O foot-and-mouth disease virus strains in my country and other susceptible viruses of cloven-hoofed animals.

[0092] Example 7: Sensitivity test of cELISA antibody detection method:

[0093] The screened monoclonal antibody A10H2-F2 and the established cELISA antibody detection method were used to detect serially diluted standard positive sera containing foot-and-mouth disease virus Re-A / WH / 2009, AKT-III, and AF / 72 vaccine strains, validating the sensitivity of the detection method. The sensitivity test results are shown in Table 6.

[0094] Table 6. Sensitivity test results of AKT-III and AF / 72 positive sera (inhibition rate %)

[0095]

[0096] As shown in Table 6, based on the 40% inhibition rate threshold, the antibodies against Re-A / WH / 2009, AKT-Ⅲ, and AF / 72 strains can be detected in positive sera diluted to a minimum of 256, 256, and 128 times, respectively, meeting the application requirements.

[0097] Example 8: Comparison of cELISA and neutralization test (SNT) results:

[0098] The cELISA antibody detection method for type A foot-and-mouth disease virus (FMDV) based on the monoclonal antibody A10H2-F2 was used to detect the neutralizing antibody background of type A FMDV in 300 sera from pigs, cattle, and sheep. The FMDV neutralizing antibody sera were prepared and validated by the National Foot-and-Mouth Disease Laboratory, and the experiment was conducted at the Biosafety Level 3 laboratory of Zhongpu Biopharmaceutical Co., Ltd. The detection results are shown in Table 7.

[0099] Table 7. Statistical analysis of cELISA method and neutralization test (VNT) results.

[0100]

[0101] As shown in Table 7, the test results show that the positive sample compliance rate is 122 / 124×100%≈98.4%, the negative sample compliance rate is 170 / 176*100%=96.6%, and the total compliance rate is (122+170) / 300×100%≈97.3%.

[0102] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0103] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A hybridoma cell line secreting monoclonal antibodies against foot-and-mouth disease virus type A, named: Foot-and-mouth disease virus type A Mc10H2-F2 hybridoma cell line, with accession number: CGMCC No.46034; deposit address: China General Microbiological Culture Collection Center.

2. Foot-and-mouth disease type A virus-specific monoclonal antibody A10H2-F2 secreted by the foot-and-mouth disease type A virus hybridoma cell line CGMCC No.46034 as described in claim 1.

3. The application of the foot-and-mouth disease type A virus Mc10H2-F2 hybridoma cell line according to claim 1 in the detection of foot-and-mouth disease virus, characterized in that, The detection includes antigen detection or antibody detection, which can specifically detect foot-and-mouth disease type A virus antigens and antibodies, but does not react with foot-and-mouth disease type O virus and other viral antigens and antibodies.

4. The application of the foot-and-mouth disease type A virus-specific monoclonal antibody A10H2-F2 as described in claim 2 in the detection of foot-and-mouth disease virus, characterized in that, The detection includes antigen detection or antibody detection, which can specifically detect foot-and-mouth disease type A virus antigens and antibodies, but does not react with foot-and-mouth disease type O virus and other viral antigens and antibodies.

5. The application as described in claim 3 or 4, characterized in that, The foot-and-mouth disease type A virus includes strains AKT-Ⅲ, AF / 72, and Re-A / WH / 2009; the foot-and-mouth disease type O virus includes strains Mya98 / xJ / 2010, OHM / 02, HB / HK / 99, GX / 09-7, and OS / 99; and the other viruses include classical swine fever virus, porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus, porcine circovirus, pseudorabies virus, bluetongue virus, bovine viral diarrhea virus, poxvirus, and akaban virus.

6. A method for preparing a hybridoma cell line secreting monoclonal antibodies against type A foot-and-mouth disease virus, characterized in that, Includes the following steps: Prokaryotic expression and purification of S1 and A type foot-and-mouth disease virus VP1 proteins; S2. The purified VP1 protein obtained in S1 was used as an antigen to immunize mice. After immunization, the spleen of the mice was removed and fused with myeloma cells to obtain aggregated hybridoma cells. S3. Initial screening: When the clustered hybridoma cell clones described in S2 grow and occupy more than 20% of the bottom area of ​​the well, the initial screening is performed using an ELISA method with Re-A / WH / 2009 strain inactivated virus as the coating antigen. OD450 ≥ 0.4 is judged as a positive clone, and the positive clones are transferred to a 24-well culture plate for further culture. S4. Second screening: When the positive clone hybridoma cells cultured in S3 grow to a density of 50% to 80%, a second screening is performed. The supernatant of each well is used to inactivate the virus with Re-A / WH / 2009 strain and to detect the antigen coated with BHK-21 cells containing 10% serum in an indirect ELISA plate. Hybridoma cell lines that can react with a mixture of serum and BHK-21 cells are eliminated, and hybridoma cell lines that can only react with foot-and-mouth disease virus are expanded to a larger scale. S5. Third Screening: When the hybridoma cells expanded from S4 grow to a density of 50%–80%, a third screening is performed, namely competitive ELISA screening. Standard positive sera of type A AKT-Ⅲ, AF72, Re-A / WH / 2009 strains and type O Mya98 / xJ / 2010, OHM / 02, HB / HK / 99, GX / 09-7, OS / 99 strains and hybridoma cell supernatant are used to competitively react with the coated A / WH / 2009 inactivated virus. Hybridoma cell lines that exhibit competitive activity with antibodies against all the above type A strains (inhibition rate ≥60%), while showing no or low competitive activity with all type O strains (inhibition rate ≤40%), are selected. These hybridoma cell lines secreting type A foot-and-mouth disease virus monoclonal antibodies are obtained.

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