CATHAY type O foot-and-mouth disease virus monoclonal antibody and its preparation kit and application

Abstract

The present invention provides monoclonal antibodies 4B12 and 10G4 that specifically bind to CATHAY-type O foot-and-mouth disease virus antigens. A double-antibody sandwich kit, prepared using monoclonal antibody 4B12 as the coating antibody and enzyme-labeled monoclonal antibody 10G4 as the detection antibody, can quantitatively detect the content of CATHAY-type O foot-and-mouth disease virus virus-like particles, whether in single, multivalent, or combination vaccines, or in semi-finished or finished vaccines.

Description

Technical Field

[0001] The present invention relates to a monoclonal antibody against type O foot-and-mouth disease virus, specifically a monoclonal antibody against type CATHAY type O foot-and-mouth disease virus, a hybridoma cell secreting the monoclonal antibody, and a double-antibody sandwich detection kit containing the monoclonal antibody and its application, belonging to the field of biotechnology. Background Art

[0002] Foot-and-mouth disease (FMD) is a febrile, acute, and highly contagious disease of even-toed ungulates caused by the FMD virus. The disease spreads rapidly and has a high incidence rate. Its outbreaks and prevalence have caused significant economic losses to the global livestock industry. The World Organization for Animal Health (OIE) and the Food and Agriculture Organization of the United Nations (FAO) classify it as a Category A highly contagious disease, and my country ranks it first among Category I infectious diseases.

[0003] There are seven serotypes of foot-and-mouth disease virus (FMDV) (A, O, C, Asia I, SAT1, SAT2, and SAT3). These serotypes are highly susceptible to mutation, and there is no cross-protection between the serotypes. In my country, FMDV types O and A are predominantly prevalent. Vaccination is an effective measure to control the disease and protect livestock from harm.

[0004] Currently, vaccines used to prevent foot-and-mouth disease primarily consist of inactivated whole-virus vaccines and synthetic peptide vaccines. However, incomplete inactivation of whole-virus vaccines poses biosafety risks. Virus-like particles (VLPs) are virus-like particles that can autonomously package into viral capsid structures when expressed in vitro and / or in vivo. They are pseudoviruses with a viral capsid structure but lack the ability to replicate. VLP vaccines can effectively stimulate the body's ability to resist infection, and vaccines designed based on VLPs are an ideal vaccine format. Therefore, the development of safe and effective VLP-based subunit vaccines is a current research hotspot.

[0005] Currently, the predominant strains of FMD-O virus are topologically based on the SEA and CATHAY types. Therefore, the development of FMD-O virus-like particle subunit vaccines is also primarily based on these topological types. The most critical technical issue during pilot testing and production is the accurate quantification of protein content in subunit vaccine semi-finished and finished products, particularly the identification of individual proteins after mixing multiple proteins. Currently, commonly used protein quantification methods include the BCA assay, Bradford assay, and Lowry assay, but these methods are limited to protein quantification and cannot distinguish the content of different proteins in a mixture of multiple proteins.

[0006] Therefore, there is an urgent need for a method for quantitatively detecting the content of foot-and-mouth disease type O virus protein, which can not only detect the content of foot-and-mouth disease type O virus protein, but also detect the content of foot-and-mouth disease type O virus protein from a mixture of multiple proteins. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention provides a pair of monoclonal antibodies that specifically bind to the Cathay-type O foot-and-mouth disease virus, as well as a double-antibody sandwich detection kit containing this monoclonal antibody pair and its application. The double-antibody sandwich ELISA method, developed based on monoclonal antibody technology, can specifically detect antigenic proteins and accurately and effectively quantify protein content, providing support for the development of viral subunit vaccines.

[0008] The present invention relates to a monoclonal antibody that specifically binds to CATHAY type O foot-and-mouth disease virus-like particles.

[0009] The present invention also relates to a kit for detecting CATHAY-type O-type foot-and-mouth disease virus-like particles with high sensitivity, good specificity and good repeatability.

[0010] The present invention also relates to a detection kit for CATHAY-type O-type foot-and-mouth disease virus-like particles with a long shelf life.

[0011] The present invention relates to a kit capable of detecting the content of foot-and-mouth disease CATHAY type O virus protein from a mixture of multiple proteins.

[0012] The invention relates to a test kit capable of detecting the content of foot-and-mouth disease CATHAY type O virus protein in a vaccine semi-finished product and a vaccine finished product.

[0013] The present invention also relates to a single-chain antibody having good reaction characteristics with CATHAY type O type foot-and-mouth disease virus-like particles. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The standard curve was prepared by diluting the standard sample with sample diluent in a 2-fold gradient and repeating the test 3 times. The results showed that the standard sample of the kit had a good linear relationship in the range of 7.8μg / ml to 1000μg / ml. 2 >0.99; detection sensitivity is 7.8μg / ml. DETAILED DESCRIPTION

[0015] definition

[0016] Foot-and-mouth disease virus (FMDV) belongs to the Picornaviridae family and the Orthomavirus genus. There are seven serotypes: O, A, C, SAT1, SAT2, SAT3 (South African FMDV types 1, 2, and 3), and Asia1 (Asia1). There is no cross-protection between the types, and each type contains multiple subtypes. At the center of the virus lies a single-stranded, positive-sense RNA, consisting of approximately 8,000 bases, which serves as the basis for infection and inheritance. The surrounding proteins determine the virus's antigenicity, immunogenicity, and serological reactivity. The viral capsid is a symmetrical icosahedron. FMDV is the causative agent of foot-and-mouth disease, a highly contagious disease of even-toed ungulates. The Office International des Epizooties (OIE) lists FMD as the first on its "List of Category A Animal Infectious Diseases." China classifies it as a "Class I Infectious Disease for Animal Quarantine at Entry." Prevention and control of FMD in my country primarily involves vaccination, and animals infected with FMD are culled.

[0017] The term "virus-like particles (VLPs)" refers to particles assembled from one or more viral structural proteins, which have external structures and antigenicity similar to viral particles but do not contain viral genes.

[0018] The term "CATHAY-type O-type foot-and-mouth disease virus-like particles" refers to virus-like particles assembled from structural proteins encoded by the CATHAY-type gene sequence of O-type foot-and-mouth disease virus. For detailed preparation, please refer to Chinese patent CN111840533A.

[0019] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible spontaneous mutations. Thus, the modifier "monoclonal" indicates that the antibody is not a mixture of discrete antibodies. Preferably, the monoclonal antibody includes monovalent or single-chain antibodies, diabodies, chimeric antibodies, humanized antibodies, derivatives, functional equivalents, and homologs of the aforementioned antibodies, as well as antibody fragments and any polypeptide containing an antigen-binding domain. Antibodies are any specific binding factor comprising a binding domain with the desired specificity, and thus, this term encompasses antibody fragments, derivatives, humanized antibodies, functional equivalents, and homologs thereof, as well as any polypeptide containing an antigen-binding domain, whether natural or synthetic. Examples of antibodies are immunoglobulin subtypes (e.g., IgG, IgE, IgM, IgD, and IgA) and their subtypes and subclasses; fragments comprising an antigen-binding domain, such as Fab, scFv, Fv, dAb, Fd; and diabodies. Chimeric molecules or equivalents comprising an antigen binding domain fused to another polypeptide are also included. The cloning and expression of chimeric antibodies are described in EP.A.0120694 and EP.A.0125023. Antibodies can be modified in many ways, and recombinant DNA techniques can be used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques can include introducing DNA encoding the immunoglobulin variable region or complementarity determining regions (CDRs) of an antibody into the constant region or constant region plus framework region of a different immunoglobulin, as described in EP.A.184187, GB2188638A, or EP.A.239400. Hybridoma cells or other cells producing antibodies can also be subjected to genetic mutations or other changes that may or may not alter the binding specificity of the produced antibody. The "monoclonal antibodies" used in the present invention can also be produced using the hybridoma method, as the DNA sequence encoding the murinized antibody of the present invention can be obtained using conventional methods familiar to those skilled in the art, such as artificially synthesizing a nucleotide sequence based on the amino acid sequence disclosed in the present invention or amplifying it using PCR. Recombinant DNA methods can also be used, and the sequence can be linked into a suitable expression vector using various methods well known in the art. Finally, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention, and then purified using conventional separation and purification methods well known to those skilled in the art to obtain the monoclonal antibodies of the present invention. Antibodies comprise a geometric structure of polypeptide chains linked together by disulfide bridges. Two polypeptide backbones, called light chains and heavy chains, constitute all major structural classes (isotypes) of antibodies. Both heavy and light chains can be further divided into subregions called variable regions and constant regions. The heavy chain includes a single variable region and three different constant regions, while the light chain includes a single variable region (different from the variable region of the heavy chain) and a single constant region (different from the constant region of the heavy chain). The variable regions of the heavy and light chains are responsible for the binding specificity of the antibody.

[0020] The term "heavy chain variable region" refers to a polypeptide having a length of 110 to 125 amino acids, the amino acid sequence of which corresponds to the heavy chain amino acid sequence of the monoclonal antibody of the present invention starting from the N-terminal amino acid of the heavy chain. Similarly, the term "light chain variable region" refers to a polypeptide having a length of 95 to 115 amino acids, the amino acid sequence of which corresponds to the light chain amino acid sequence of the monoclonal antibody of the present invention starting from the N-terminal amino acid of the light chain. It is obvious to those skilled in the art that, based on the amino acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody specifically disclosed in the present invention, one or more amino acids can be added, deleted, substituted, etc. by conventional genetic engineering and protein engineering methods to obtain conservative variants while still being able to maintain specific binding to canine rotavirus. The monoclonal antibodies of the present invention also include active fragments or conservative variants thereof.

[0021] The term "conservative variant" refers to a variant that has substantially retained the characteristics of its parent, such as basic immunological biological properties, structural properties, regulatory properties or biochemical properties. Generally, the amino acid sequence of the conservative variant of a polypeptide is different from that of the parent polypeptide, but the difference is limited so that the sequence of the parent polypeptide is very similar to the conservative variant overall and is identical in many regions. The difference in the conservative variant and the parent polypeptide amino acid sequence can be, for example, the replacement, addition and deletion of one or more amino acid residues and any combination thereof. The amino acid residues replaced or inserted may or may not be encoded by the genetic code. The conservative variant of a polypeptide can occur naturally, or it can be a non-naturally occurring variant. The non-naturally occurring conservative variant of a polypeptide can be produced by mutagenesis techniques or direct synthesis.

[0022] The term "genetically engineered antibody", also known as recombinant antibody, refers to an antibody molecule that is expressed by transfecting appropriate receptor cells after the antibody-encoding genes have been processed, modified and reassembled according to different needs using recombinant DNA and protein engineering technologies.

[0023] The term "single chain antibody fragment" (scFv) refers to an antibody composed of the heavy chain variable region and the light chain variable region of the antibody connected by a short peptide (linker) of 15 to 20 amino acids.

[0024] The term "chimeric monoclonal antibody" refers to a human-mouse hybrid antibody created by replacing the mouse constant region with a human constant region while retaining the variable region sequence of the mouse monoclonal antibody. This hybrid antibody is rapidly developed and can significantly reduce the immunogenicity of the heterologous antibody while retaining nearly all of the specificity and affinity of the parental mouse monoclonal antibody.

[0025] The term "degenerate sequence" refers to the phenomenon in molecular biology that the same amino acid has two or more codons, which is called codon degeneracy. Such a sequence is called a degenerate sequence.

[0026] The term "semi-finished product" refers to the inactivated virus (bacteria) liquid or the virus (bacteria) liquid harvested from the attenuated vaccine or the protein expressed by genetic engineering.

[0027] The term "finished product" refers to a product prepared by mixing semi-finished products and adjuvants in appropriate proportions according to production steps. Detailed Description of the Invention

[0029] The present invention relates to a variable region sequence of a monoclonal antibody 4B12 that specifically binds to a CATHAY-type O foot-and-mouth disease virus antigen, wherein the heavy chain variable region of the monoclonal antibody 4B12 is encoded by SEQ.ID No.1 or a degenerate sequence thereof; and the light chain variable region of the monoclonal antibody 4B12 is encoded by SEQ.ID No.2 or a degenerate sequence thereof.

[0030] The present invention relates to an antibody or antibody fragment that specifically binds to a CATHAY-type O-type foot-and-mouth disease virus antigen, wherein the heavy chain variable region of the antibody or antibody fragment is encoded by SEQ.ID No.1 or a degenerate sequence thereof, and the light chain variable region of the antibody or antibody fragment is encoded by SEQ.ID No.2 or a degenerate sequence thereof; the antibody is a monoclonal antibody or a genetically engineered antibody; wherein the genetically engineered antibody includes a single-chain antibody, a chimeric monoclonal antibody, and a reshaped monoclonal antibody; and the antibody or antibody fragment specifically binds to a CATHAY-type O-type foot-and-mouth disease virus-like particle.

[0031] As one embodiment of the present invention, the antibody is monoclonal antibody 4B12, the heavy chain variable region of the monoclonal antibody 4B12 is encoded by SEQ.ID No.1 or its degenerate sequence, and the light chain variable region of the monoclonal antibody 4B12 is encoded by SEQ.ID No.2 or its degenerate sequence.

[0032] The CATHAY O-type foot-and-mouth disease virus monoclonal antibody 4B12 of the present invention is obtained by screening after immunization with CATHAY O-type foot-and-mouth disease virus-like particles, and the ELISA titer is 1:819200.

[0033] The present invention also relates to a hybridoma cell line 4B12, which secretes the monoclonal antibody 4B12.

[0034] The present invention also relates to a variable region sequence of a monoclonal antibody 10G4 that specifically binds to a CATHAY type O foot-and-mouth disease virus antigen, wherein the heavy chain variable region of the monoclonal antibody 10G4 is encoded by SEQ.ID No.3 or a degenerate sequence thereof; and the light chain variable region of the monoclonal antibody 10G4 is encoded by SEQ.ID No.4 or a degenerate sequence thereof.

[0035] The present invention relates to an antibody or antibody fragment that specifically binds to a CATHAY-type O-type foot-and-mouth disease virus antigen, wherein the heavy chain variable region of the antibody or antibody fragment is encoded by SEQ.ID No.3 or a degenerate sequence thereof, and the light chain variable region of the antibody or antibody fragment is encoded by SEQ.ID No.4 or a degenerate sequence thereof; the antibody is a monoclonal antibody or a genetically engineered antibody; wherein the genetically engineered antibody includes a single-chain antibody, a chimeric monoclonal antibody, and a reshaped monoclonal antibody; and the antibody or antibody fragment specifically binds to a CATHAY-type O-type foot-and-mouth disease virus-like particle.

[0036] As one embodiment of the present invention, the antibody is monoclonal antibody 10G4, the heavy chain variable region of the monoclonal antibody 10G4 is encoded by SEQ.ID No.3 or its degenerate sequence, and the light chain variable region of the monoclonal antibody 10G4 is encoded by SEQ.ID No.4 or its degenerate sequence.

[0037] The CATHAY O-type foot-and-mouth disease virus monoclonal antibody 10G4 of the present invention is obtained by screening after immunization with CATHAY O-type foot-and-mouth disease virus-like particles, and the ELISA titer is 1:819200.

[0038] The present invention also relates to a hybridoma cell line 10G4, which secretes the monoclonal antibody 10G4.

[0039] The hybridoma cell lines 4B12 and 10G4 can effectively secrete monoclonal antibodies 4B12 and 10G4, respectively. The secreted monoclonal antibodies 4B12 and 10G4 are both of high purity.

[0040] The present invention also relates to the application of the antibody or antibody fragment or monoclonal antibody, wherein the application is epitope identification research and CATHAY type O foot-and-mouth disease virus antigen reactivity research.

[0041] The present invention also relates to a kit for detecting cathay type o foot-and-mouth disease virus antigens, wherein the kit comprises: a supporting medium coated with the monoclonal antibody 4B12, the supporting medium being blocked with a blocking solution after being coated with the monoclonal antibody 4B12, an enzyme-labeled monoclonal antibody 10G4; a 20× concentrated washing solution, the 20× concentrated washing solution being a 0.1M PBS solution with a pH of 7.4 containing 10% v / v Tween 20; a standard substance, the standard substance being cathay type o foot-and-mouth disease virus-like particles; a color developing solution, the color developing solution comprising a color developing solution A and a color developing solution B, the color developing solution A being an aqueous solution containing 14.7 g / L disodium hydrogen phosphate, 9.3 g / L citric acid, and 0.3 g / L urea peroxide, and the color developing solution B being an aqueous solution of 0.2 g / L tetramethylbenzenediamine (TMB) and 10% v / v anhydrous ethanol; and a stop solution, the stop solution being a 2M H2SO4 solution.

[0042] As an embodiment of the present invention, the support medium coated with monoclonal antibody 4B12 is a microtiter plate, the working concentration of the monoclonal antibody 4B12 is 0.05 μg / well to 0.1 μg / well, and the titer of the enzyme-labeled monoclonal antibody 10G4 is 1:40 to 1:80; preferably, the working concentration of the monoclonal antibody 4B12 is 0.1 μg / well, the titer of the enzyme-labeled monoclonal antibody 10G4 is 1:40, and the enzyme is horseradish peroxidase; the blocking solution is 0.1M PBS at pH 7.4 containing 1% w / v soy protein, 1% w / v sucrose, and 0.05% v / v Tween 20; the standard is 1 mg / ml CATHAY type O foot-and-mouth disease virus-like particles; the kit also includes a sample diluent, which is a pH 7.4 solution containing 0.5% to 2% w / v sucrose, 1% w / v EDTA, and 0.004% w / v AW dye. 7.4 of 0.1 M PBS solution.

[0043] The coating concentration of the monoclonal antibody 4B12 can be 0.05 μg / well, 0.051 μg / well, 0.052 μg / well, 0.053 μg / well, 0.054 μg / well, 0.055 μg / well, 0.056 μg / well, 0.057 μg / well, 0.058 μg / well, 0.059 μg / well, 0.06 μg / well, 0.06 1μg / well, 0.062μg / well, 0.063μg / well, 0.064μg / well, 0.065μg / well, 0.066μg / well, 0.067μg / well, 0.068μg / well, 0.069μg / well, 0.070μg / well, 0.071μg / well, 0.072μg / well, 0.073μg / well, 0.074μ g / well, 0.075 μg / well, 0.076 μg / well, 0.077 μg / well, 0.078 μg / well, 0.079 μg / well, 0.080 μg / well, 0.081 μg / well, 0.082 μg / well, 0.083 μg / well, 0.084 μg / well, 0.085 μg / well, 0.086 μg / well, 0.087 μg / well, 0.088μg / well, 0.089μg / well, 0.090μg / well, 0.091μg / well, 0.092μg / well, 0.093μg / well, 0.094μg / well, 0.095μg / well, 0.096μg / well, 0.097μg / well, 0.098μg / well, 0.099μg / well, 0.10μg / well.

[0044] The titer of the enzyme-labeled reagent, that is, the titer of the enzyme-labeled monoclonal antibody 10G4, can be 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, or 1:80.

[0045] The double-antibody sandwich detection kit of the present invention can detect CATHAY type O type foot-and-mouth disease virus, can accurately quantify the content of CATHAY type O type foot-and-mouth disease virus antigen in a vaccine containing CATHAY type O type foot-and-mouth disease virus subunits, and provides support for the inspection of vaccine semi-finished products and finished products.

[0046] The present invention ensures higher detection sensitivity by selecting the fixed content of monoclonal antibody 4B12 and the potency of enzyme-labeled monoclonal antibody 10G4.

[0047] The present invention also relates to a method for preparing the kit, wherein the method comprises: step (1) diluting the monoclonal antibody 4B12 with a carbonate buffer solution of pH 9.6 and coating the solution on a microtiter plate; step (2) blocking the microtiter plate coated with the monoclonal antibody 4B12 in step (1) with the blocking solution; step (3) preparing the sample diluent, the enzyme-labeled monoclonal antibody 10G4, the 20× concentrated washing solution, the standard, the color developing solution, and the stop solution; step (4) assembling the blocked microtiter plate in step (2), the sample diluent in step (3), the 20× concentrated washing solution, the color developing solution, and the stop solution into a kit A box, and assembling the standard in step (3) into a kit B box.

[0048] As an embodiment of the present invention, in step (1), a support medium coated with monoclonal antibody 4B12 is prepared by using carbonate buffer (pH 9.6) and a concentration of monoclonal antibody 4B12 of 0.05 μg / well to 0.1 μg / well, coating 100 μl / well in a microtiter plate at 2 to 8°C for 12 to 16 hours, washing with a washing solution and patting the reaction plate dry; blocking with a blocking solution at 2 to 8°C for 12 to 16 hours, and vacuum packaging after drying.

[0049] As an embodiment of the present invention, the step (2) is to prepare a sample diluent, a washing solution, a color developing solution, and a stop solution, and to subpackage them.

[0050] As an embodiment of the present invention, in the step (3) of preparing the enzyme-labeled reagent, the monoclonal antibody 10G4 is enzyme-labeled and then diluted with the sample diluent to 1:40 to 1:80.

[0051] As an embodiment of the present invention, in step (4), a standard is prepared by diluting the CATHAY-type foot-and-mouth disease virus-like particles to 1 mg / ml with the sample diluent and packaging;

[0052] As an embodiment of the present invention, in step (5), the supporting medium prepared in step (1), the sample diluent, washing solution, developing solution, stop solution prepared in step (2), and the enzyme-labeled reagent prepared in step (3) are used as a kit A, and the storage conditions of the kit A are 2 to 8°C; the standard prepared in step (4) is used as a kit B, and the storage conditions of the kit B are -20°C.

[0053] As an embodiment of the present invention, the concentration of the monoclonal antibody 4B12 in step (1) is 0.1 μg / well.

[0054] As an embodiment of the present invention, the blocking solution in step (1) is 0.1M PBS with a pH of 7.4 containing 1% w / v soy protein, 1% w / v sucrose, and 0.05% v / v Tween 20.

[0055] As an embodiment of the present invention, the sample diluent in step (2) is a 0.1M PBS solution with a pH of 7.4 containing 0.5% to 2% w / v sucrose, 1% w / v EDTA, and 0.004% w / v AW dye; the washing solution is a 0.1M PBS solution with a pH of 7.4 containing 10% v / v Tween 20, which is diluted 20 times with purified water when used; the color developing solution includes color developing solution A and color developing solution B, the color developing solution A is a purified water mixture containing 14.7g / L disodium hydrogen phosphate, 9.3g / L citric acid, and 0.3gL urea peroxide, and the color developing solution B is a purified water mixture containing 0.2g / L tetramethylbenzyl diamine (TMB) and 100ml / L anhydrous ethanol; and the stop solution is a 2M H2SO4 solution.

[0056] As an embodiment of the present invention, the titer of the enzyme-labeled reagent in step 3) is 1:40.

[0057] The present invention also relates to the use of the kit in detecting the content of CATHAY-type O-type foot-and-mouth disease virus-like particles; wherein the content of CATHAY-type O-type foot-and-mouth disease virus-like particles is the content of CATHAY-type O-type foot-and-mouth disease virus-like particles in a single vaccine, a multivalent vaccine, or a combined vaccine.

[0058] As an embodiment of the present invention, the single vaccine, multivalent vaccine, or combined vaccine is a semi-finished vaccine or a finished vaccine.

[0059] The present invention also relates to a single-chain antibody, wherein the heavy chain variable region of the single-chain antibody is encoded by SEQ.ID No.1 or a degenerate sequence thereof, or the heavy chain variable region of the single-chain antibody is encoded by SEQ.ID No.3 or a degenerate sequence thereof; the light chain variable region of the single-chain antibody is encoded by SEQ.ID No.2 or a degenerate sequence thereof, or the light chain variable region of the single-chain antibody is encoded by SEQ.ID No.4 or a degenerate sequence thereof.

[0060] The ELISA titer of the single-chain antibody to CATHAY-type O-type foot-and-mouth disease virus is ≥1:1600, indicating that the single-chain antibody has good reaction characteristics with CATHAY-type O-type foot-and-mouth disease virus.

[0061] Beneficial effects of the present invention:

[0062] The two CATHAY-type O-type foot-and-mouth disease virus monoclonal antibodies prepared by the invention can specifically identify CATHAY-type O-type foot-and-mouth disease virus.

[0063] The kit prepared by the present invention can be used for quantitative detection of semi-finished products and finished products containing cathay type O type foot-and-mouth disease virus subunit vaccines, and is easier to conduct research and development pilot tests and production quality control.

[0064] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0065] All chemical reagents used in the examples of the present invention were of analytical grade and purchased from Sinopharm Group.

[0066] The coating solution used in the examples of the present invention is a carbonate buffer solution with a pH of 9.6. Unless otherwise specified, the solution is diluted with a phosphate buffer solution, but this embodiment does not limit the present invention in any case.

[0067] Unless otherwise specified, the experimental methods described in the present invention are all conventional methods; unless otherwise specified, the biological materials described can be obtained from commercial channels.

[0068] Example 1 Preparation and Identification of Foot-and-Mouth Disease Virus-like Particles

[0069] For the preparation and identification of CATHAY-type O-type foot-and-mouth disease virus-like particles, SEA-type O-type foot-and-mouth disease virus-like particles and A-type foot-and-mouth disease virus-like particles, please refer to Chinese patent CN111840533A.

[0070] Example 2 Preparation and Identification of Monoclonal Antibodies to CATHAY Type O Foot-and-Mouth Disease Virus

[0071] 2.1 Preparation of monoclonal antibodies against CATHAY type O foot-and-mouth disease virus

[0072] The CATHAY-type O-type foot-and-mouth disease virus-like particles prepared in Example 1 were used as immunogens to immunize 5 Balb / c mice aged 5 to 6 weeks. The immunization method was multiple subcutaneous injections on the back, with an injection volume of 50 to 100 μg per mouse. The first immunization was emulsified with an equal volume of Freund's complete adjuvant and the immunogen. After an interval of 2 weeks, the booster immunization was performed. During the booster immunization, an equal volume of Freund's incomplete adjuvant and the immunogen were emulsified, and the booster immunization was repeated twice. The antibody titer of the mouse serum was detected by an indirect ELISA method using CATHAY-type O-type foot-and-mouth disease virus-like particles (the coating amount of CATHAY-type O-type foot-and-mouth disease virus-like particles was 0.1 μg / ml, 100 μl / well). The antibody titer after three immunizations was 1:12800-1:51200 one week after the three immunizations, and 1:51200-1:204800 two weeks after the three immunizations. Mice with the highest antibody titer (i.e., 1:204800) were selected for pulse immunization, i.e., antigen was injected into the tail vein. Three days after the pulse immunization, the spleens of the mice were harvested and fused with myeloma cells. Hybridoma cells were subcloned by limiting dilution, and positive hybridoma cells were screened by indirect ELISA. A total of 8 positive hybridoma cell lines were obtained and numbered as hybridoma cell G1, hybridoma cell G2, hybridoma cell G3, hybridoma cell G4, hybridoma cell G5, hybridoma cell G6, hybridoma cell G7, and hybridoma cell G8. The cell supernatants were tested by indirect ELISA, and the antibody titers were 1:256, 1:128, 1:128, 1:512, 1:128, 1:256, 1:128, and 1:256, respectively.

[0073] Ascites fluid was prepared from the eight hybridoma cell lines described above using the mouse ascites method, and each ascites fluid was purified to obtain monoclonal antibodies G1, G2, G3, G4, G5, G6, G7, and G8. Sodium acetate buffer was first added to the ascites fluid to adjust the pH to 5.0, and the fluid was stirred at 2-8°C for 30 minutes, during which time octanoic acid was slowly added. The fluid was allowed to stand for 2 hours at 2-8°C, and then centrifuged at 10,000 rpm for 30 minutes. The supernatant was then collected. PBS solution was added, and saturated ammonium sulfate solution was added over 30 minutes at 2-8°C to a final concentration of 0.277 g / ml. The fluid was allowed to stand for 1 hour. Centrifuged at 10,000 rpm for 30 minutes, the supernatant discarded, and the precipitate dissolved in PBS (1 / 5 of the initial ascites fluid volume). The fluid was dialyzed against 100 volumes of PBS at 4°C overnight. Insoluble matter was removed by centrifugation at 4°C, and the supernatant was the purified antibody.

[0074] 2.2 Identification of monoclonal antibodies against CATHAY type O foot-and-mouth disease virus

[0075] 2.2.1 Monoclonal Antibody Purity Identification and Protein Content Determination

[0076] The results of SDS-PAGE gel electrophoresis showed that the purity of each purified monoclonal antibody was greater than 90%.

[0077] The concentrations of the purified monoclonal antibodies were determined using a BCA protein concentration assay kit, which were 1.9 mg / ml, 3.6 mg / ml, 3.1 mg / ml, 3.3 mg / ml, 3.1 mg / ml, 3.7 mg / ml and 1.1 mg / ml, 3.5 mg / ml, respectively. The yields of two monoclonal antibodies were low after purification.

[0078] 2.2.2 Identification of monoclonal antibody types and subclasses

[0079] The subtypes of 8 monoclonal antibodies were identified using a monoclonal antibody subclass identification kit. The results showed that the heavy chain subclasses of the 8 monoclonal antibodies were IgG2b, IgG1, IgG2a, IgG1, IgG2a, IgG2b, IgG2b, and IgG2b, respectively, and the light chain subclasses were all kappa.

[0080] 2.2.3 Potency determination

[0081] The titers of 8 monoclonal antibodies were detected by indirect ELISA method, which were 1:102400, 1:819200, 1:819200, 1:1638400, 1:3276800, 1:819200, 1:102400, and 1:819200, respectively.

[0082] 2.2.4 Monoclonal Antibody Specificity Identification

[0083] The reactivity of 8 monoclonal antibodies with SEA-type O-type foot-and-mouth disease virus-like particles was detected by indirect ELISA. The specific operation was as follows: SEA-type O-type foot-and-mouth disease virus-like particles were diluted to 1 μg / ml with coating solution, mixed and coated onto the ELISA plate, 100 μl / well, sealed with a sealing film and allowed to stand at 2-8°C for 24 hours; the liquid in the wells was discarded, and 200 μl of blocking solution (5% w / v skim milk, pH 5.0) was added to each well. 7.4PBS), seal the plate with a sealing film and block at 37°C for 2 hours; discard the liquid in the wells, wash the plate and pat dry, add 100-fold diluted 7 monoclonal antibodies respectively, and add PBS as a negative control. The positive mouse serum after immunization with SEA type O type foot-and-mouth disease virus-like particles is a positive control, all at 50 μl / well, and act at 37°C for 1 hour; discard the liquid in the wells, wash the plate and pat dry, add 1:50000-fold diluted HRP-labeled goat anti-mouse IgG, 50 μl / well, and act at 37°C for 30 minutes; discard the liquid in the wells, wash the plate and pat dry, add color reagent A solution and B solution, 50 μl / well, mix well, and act at 37°C for 15 minutes; add stop solution, 50 μl / well, and read OD 450nm-630nmThe test was established when the negative control OD value was <0.2 and the positive control OD value was ≥1.0. A positive result was determined when the S / N (sample OD value / negative control OD value) was ≥2.1, and a negative result was determined when the S / N (sample OD value / negative control OD value) was <2.1. The test results showed that monoclonal antibodies G1, G2, G4, G5, G7, and G8 were reactive with SEA-type O-type FMDV-like particles, while monoclonal antibodies G3 and G6 were not reactive with SEA-type O-type FMDV-like particles.

[0084] The 8 monoclonal antibodies were tested using the foot-and-mouth disease virus type A antibody liquid phase blocking ELISA detection kit according to its instructions. The antibody titers were all less than 1:8, indicating that none of them reacted with foot-and-mouth disease virus type A.

[0085] The specificity of eight monoclonal antibodies was evaluated using common swine viruses. IFA was used to detect the antibodies. The assay was considered valid when the healthy cell control wells showed no fluorescence and the positive control wells showed yellow-green fluorescence. Wells with infected cells exhibited yellow-green fluorescence, which was considered positive; wells with infected cells exhibited no yellow-green fluorescence, which was considered negative. Results: None of the eight monoclonal antibodies reacted with pseudorabies virus (PRV), porcine parvovirus (PPV), classical swine fever virus (CSFV), porcine circovirus type 2 (PCV2), or porcine reproductive and respiratory syndrome virus (PRRSV).

[0086] These results indicate that monoclonal antibodies G1, G2, G4, G5, G7, and G8 are reactive with both CATHAY-type O-type FMDV-like particles and SEA-type O-type FMDV-like particles. Monoclonal antibodies G3 and G6 are specific only for CATHAY-type O-type FMDV. Therefore, monoclonal antibodies G3 and G6 were selected for the quantitative analysis of CATHAY-type O-type FMDV-like particles. These antibodies were designated 4B12 and 10G4.

[0087] Example 3 Preparation and identification of enzyme-labeled antibodies

[0088] 3.1 Preparation

[0089] Monoclonal antibodies 4B12 and 10G4 were labeled with horseradish peroxidase (HRP) using a modified sodium periodate method. 20 mg of HRP was dissolved in 1 ml of ultrapure water. 1 ml of freshly prepared NaIO4 solution (30 mg NaIO4 dissolved in 1 ml of ultrapure water) was added, mixed, and incubated at 2-8°C in the dark for 30 minutes. 40 μl of ethylene glycol was added to this solution and incubated at 2-8°C in the dark for 30 minutes. 1 mg of each purified monoclonal antibody was added to 100 μl of this mixture, and the mixture was added to a dialysis bag, mixed, and dialyzed against CB buffer for 6 hours. The entire procedure must be performed in the dark. Transfer the dialyzed mixture to a 1.5ml EP tube and add 10μl of freshly prepared NaBH4 solution (10mg NaBH4 dissolved in 1ml ultrapure water). Incubate at room temperature for 2 hours, mixing every 30 minutes. Add an equal volume of saturated ammonium sulfate, mix thoroughly, and incubate at 2-8°C for 15 minutes. Centrifuge at 12,000 rpm for 10 minutes and discard the supernatant. Resuspend the precipitate in an equal volume of PBS and glycerol (V:V = 1:1) as the purified antibody. This is the enzyme-labeled monoclonal antibody and store at -20°C until needed.

[0090] 3.2 Identification

[0091] Appearance: At room temperature, it is a reddish-brown liquid with no flocculent precipitation.

[0092] Quality evaluation: Use a UV spectrophotometer to detect the absorbance value A of the enzyme-labeled antibody at 403nm and 280nm. Calculate the corresponding enzyme parameters according to the formula:

[0093] Enzyme amount (mg / ml) = A 403nm ×0.4×dilution factor.

[0094] IgG amount (mg / ml) = (A 280nm -A 403nm ×0.3)×0.62×dilution factor.

[0095] Molar ratio (E / P) = enzyme amount × 4 / IgG amount.

[0096] Marking rate = A 403nm / A 280nm .

[0097] After absorbance detection and calculation, the specific results are shown in Table 1. Table 1 shows the antibody enzyme labeling situation and demonstrates the enzyme labeling quality.

[0098] Table 1 Quality evaluation of enzyme-labeled antibodies

[0099] Antibody <![CDATA[A 403nm ]]> <![CDATA[A 280nm ]]> Enzyme amount IgG amount Molar ratio Marking rate 4B12 0.431 0.494 1.724 2.261 3.05 0.873 10G4 0.413 0.457 1.652 2.065 3.0 0.904

[0100] 3.3 Enzyme-labeled antibody activity detection

[0101] The CATHAY-type O-type foot-and-mouth disease virus-like particles prepared in Example 1 were used as coating agents and coated on a microplate at 1 μg / ml, 100 μl / well, and then blocked with pH 7.4 PBS containing 5% w / v skim milk at 37°C for 2 hours; the liquid in the wells was discarded, the plate was washed and patted dry. The enzyme-labeled monoclonal antibodies 4B12 and 10G4 were diluted 10,000, 20,000, 40,000, 80,000, 160,000, 320,000, and 640,000 times with pH 7.4 PBS, respectively, at 50 μl / well. PBS was also added as a negative control, at 50 μl / well, and incubated at 37°C for 1 hour; the liquid in the wells was discarded, the plate was washed and patted dry, and color developer solution A and solution B were added, at 50 μl / well, respectively, mixed, and incubated at 37°C for 15 minutes; stop solution was added, at 50 μl / well, and the OD was read. 450nm-630nm Results were determined as follows: a S / N (sample OD value / negative control OD value) ≥ 2.1 was considered positive, and a S / N (sample OD value / negative control OD value) < 2.1 was considered negative. The reciprocal of the maximum dilution factor for a sample S / N value ≥ 2.1 was used as the titer for that sample. Results: The ELISA titers of monoclonal antibodies 4B12 and 10G4 were 1:40,000 and 1:80,000, respectively.

[0102] 3.4 Variable region sequence determination of monoclonal antibodies 4B12 and 10G4

[0103] Based on the sequence characteristics of mouse monoclonal antibodies, the heavy chain variable region primer sequences were designed:

[0104] P1: 5'-ACTAGTCGACATGAAATGCTCGTGGRTYATSAACTT-3'

[0105] P2: 5'-ACTAGTCGACATGAAATGCAGCTGGRTYAT-3'

[0106] Design light chain variable region primer sequences:

[0107] P3: 5'-ACTAGTCGACATGGTYGTYATVTCCTTGCT-3'

[0108] P4: 5'-ACTAGTCGACATGGGCWTCAAGATGRAGTCACAKW-3'

[0109] Hybridoma cells 4B12 and 10G4 were cultured and collected, and RNA was extracted and reverse transcribed as a template. The variable region sequences were amplified using the aforementioned primers, and the amplified products were sent to Suzhou Genewise Biotechnology Co., Ltd. for sequencing. Results: The nucleotide sequences of the heavy and light chain variable regions of monoclonal antibody 4B12 are shown in SEQ.ID No. 1 and SEQ.ID No. 2, respectively. The nucleotide sequences of the heavy and light chain variable regions of monoclonal antibody 10G4 are shown in SEQ.ID No. 3 and SEQ.ID No. 4, respectively.

[0110] Example 4 Preparation of kit and establishment of detection method

[0111] 4.1 Preparation of the kit

[0112] Coating plate: Dilute the purified monoclonal antibody prepared in Example 2 to the preferred concentration in carbonate buffer (pH 9.6, as the coating solution) and coat the plate in a microtiter plate at 100 μl / well. Incubate at 2-8°C for 12-16 hours. Wash with washing solution and pat dry the plate. Block with blocking solution (prepared as in Example 4.5) at 2-8°C for 12-16 hours. After drying, vacuum seal and store at 2-8°C until use.

[0113] Sample diluent: 0.1 M PBS solution at pH 7.4 containing 0.5% to 2% w / v sucrose, 1% w / v EDTA, and 0.004% w / v AW dye. Filter through 0.22 μm and aliquot aseptically.

[0114] Enzyme-labeled reagent: dilute the enzyme-labeled antibody prepared in Example 3 with sample diluent to an antibody titer of 1:40 to 1:80, filter through 0.22 μm, and dispense aseptically.

[0115] 20× concentrated washing solution: 0.1 M PBS solution with pH 7.4 containing 10% v / v Tween 20, filtered through 0.22 μm, sterilely aliquoted, and diluted 20-fold with purified water before use.

[0116] Standard: The CATHAY-type O foot-and-mouth disease virus-like particles prepared in Example 1 were diluted to 1 mg / ml with sample diluent, filtered through 0.22 μm, sterilely packaged, and stored at -20°C.

[0117] Chromogenic solution: including chromogenic solution A and chromogenic solution B. Dissolve 14.7 g of disodium hydrogen phosphate, 9.3 g of citric acid, and 0.3 g of urea peroxide in purified water and dilute to 1 L. Filter through 0.22 μm and aseptically aliquot to obtain chromogenic solution A. Dissolve 0.2 g of tetramethylbenzyl diamine (TMB) and 100 ml of anhydrous ethanol in purified water and dilute to 1 L. Filter through 0.22 μm and aseptically aliquot to obtain chromogenic solution B.

[0118] Stop solution: 2M H2SO4 solution.

[0119] The CATHAY O-type foot-and-mouth disease virus-like particle assay kit includes Box A and Box B. Assemble Box A with the coated plate, sample diluent, enzyme-labeled reagent, 20× concentrated wash buffer, colorimetric solution A, colorimetric solution B, and stop buffer and store at 2–8°C until use. The standard is in Box B, stored at -20°C.

[0120] 4.2 Establishment of kit detection method

[0121] The detection method of the kit includes the following steps:

[0122] Step 1) dilute the standard sample 2-fold, i.e., 2-fold dilution, 4-fold dilution, 8-fold dilution, 16-fold dilution, 32-fold dilution, 64-fold dilution, 128-fold dilution, and 256-fold dilution, and add 50 μl / well to the antigen-coated plate;

[0123] Step 2) Dilute the protein sample to be tested at an appropriate dilution factor, 50 μl / well, add it to the coated plate, and gently shake to mix; set up one blank control well (add 50 μl of sample diluent). Seal the plate with sealing film and incubate at 37°C for 60 minutes;

[0124] Step 3) Discard the liquid in the plate and wash the reaction plate 3-5 times, patting it dry as much as possible on the last wash;

[0125] Step 4) Add 50 μl of enzyme-labeled reagent to each well, gently shake to mix, seal the plate with sealing film, and incubate at 37°C for 30 minutes;

[0126] Step 5) Discard the liquid in the plate and wash the reaction plate 3-5 times, patting it dry as much as possible on the last wash;

[0127] Step 6) Add 50 μl of colorimetric solution A and 50 μl of colorimetric solution B to each well, shake gently to mix, and incubate at 37°C in the dark for 15 minutes;

[0128] Step 7) Add 50 μl of stop solution to each well, gently shake to mix, and measure the results within 10 minutes;

[0129] Step 8) Setting the dual wavelength to 450nm-630nm, measure the OD value of each well;

[0130] Step 9) Create a 1g-1g standard curve based on the OD value and protein content of the standard, and calculate the content of the sample to be tested based on the standard curve.

[0131] Test establishment condition: standard curve R 2The standard curve should be ≥0.99 and contain at least 5 points. Otherwise, the experiment is invalid and the test should be repeated. The working range of the standard curve is 7.8μg / ml to 1000μg / ml. The linear relationship is good within this range. The dilution factor of the sample to be tested should be selected based on this range.

[0132] Result determination: The mean of the protein content corresponding to different dilution multiples within the linear relationship range was selected as the content of the sample.

[0133] 4.3 Monoclonal Antibody Pairing Test for Kits

[0134] Enzyme-labeled antibodies 4B12 and 10G4 prepared in Example 3 were used with purified monoclonal antibodies 4B12 and 10G4 prepared in Example 2 to prepare coated plates according to Example 4.1 (coating monoclonal antibody concentration: 0.1 μg / well, enzyme-labeled antibody titer: 1:40). The plates were paired according to Table 2 to form test kits. The dilutions of the CATHAY-type O foot-and-mouth disease virus-like particles prepared in Example 1 were tested using the detection method described in Example 4.2. Suitable pairing systems were screened based on the sensitivity of the assay. The results are shown in Table 2. The highest sensitivity (15.6 μg / ml) was achieved when monoclonal antibody 4B12 was used as the coating monoclonal antibody and monoclonal antibody 10G4 was used as the enzyme-labeled antibody. Therefore, coating antibody 4B12 and enzyme-labeled antibody 10G4 were used in subsequent studies.

[0135] Table 2 Sensitivity test results of monoclonal antibody pairing test

[0136]

[0137] Note: “ / ” means no pairing is performed.

[0138] 4.4 Optimization of the concentration of coated monoclonal antibody and enzyme-labeled antibody in the kit

[0139] The coating monoclonal antibody working concentrations (0.25 μg / well, 0.1 μg / well, 0.05 μg / well, and 0.025 μg / well) and the enzyme-labeled antibody titers (1:80, 1:40, 1:20, and 1:10) shown in Table 3 were used to test CATHAY-type O virus-like particles at a 200 μg / ml dilution in triplicate. Sample dilutions were used as negative controls in duplicate in two wells. Appropriate coating monoclonal antibody and enzyme-labeled antibody working concentrations were selected based on the P / N values. Results (see Table 3) showed that the P / N values were higher when the coating antibody working concentration was 0.05 μg / well to 0.1 μg / well and the enzyme-labeled antibody titer was 1:40 to 1:80, indicating that kits were suitable for preparation. The kit prepared with a coating antibody working concentration of 0.1 μg / well and an enzyme-labeled antibody titer of 1:40 achieved the best results.

[0140] Table 3 Results of optimization test on working concentration of coated monoclonal antibody and enzyme-labeled antibody (P / N value)

[0141]

[0142] 4.5 Optimization of blocking solution in the kit

[0143] Kits were prepared using different blocking solution formulations (see Table 4) while maintaining other conditions unchanged. The kits were then subjected to sensitivity and specificity testing (see Example 5). The results (Table 4) showed that the highest detection sensitivity was achieved when the blocking solution was 0.1 M PBS, pH 7.4, containing 1% w / v soy protein, 1% w / v sucrose, and 0.05% v / v Tween 20.

[0144] Table 4 Blocking solution optimization results

[0145]

[0146] Based on this, the present invention uses a coating antibody working concentration of 0.1 μg / well, an enzyme-labeled antibody titer of 1:40, and a blocking solution of 0.1 M PBS at pH 7.4 containing 1% w / v soy protein, 1% w / v sucrose, and 0.05% v / v Tween 20. The kit prepared according to Example 4.1 is used as the optimal kit for subsequent detection.

[0147] Example 5 Application of the kit

[0148] 5.1 Linear range of the standard curve and sensitivity test of the kit

[0149] The CATHAY type O virus-like particle standard (1 mg / ml) was diluted with sample diluent in a 2-fold gradient (corresponding to protein content of 1000 μg / ml, 500 μg / ml, 250 μg / ml, 125 μg / ml, 62.5 μg / ml, 31.3 μg / ml, 15.6 μg / ml, 7.8 μg / ml, and 3.9 μg / ml), and the test was repeated three times. Results (see Figure 1 ): The standard product of the kit has a good linear relationship in the range of 7.8μg / ml to 1000μg / ml, R 2 >0.99; detection sensitivity is 7.8μg / ml.

[0150] 5.2 Specificity detection

[0151] According to the method of Example 1, three batches of SEA-type O virus-like particles and three batches of type A virus-like particles were prepared, and the prepared kits were used for detection. The results (see Table 5) showed that the OD values of the three batches of SEA-type O virus-like particles and the three batches of type A virus-like particles were basically consistent with the blank control values, indicating that the kit had good specificity.

[0152] Table 5 Kit specificity detection results

[0153]

[0154] 5.3 Repeatability test

[0155] The CATHAY-type O foot-and-mouth disease virus-like particles prepared in Example 1 were diluted with sample diluent to 500 μg / ml, 200 μg / ml, 80 μg / ml, and 40 μg / ml, respectively. Inter- and intra-batch testing was repeated five times. The results (see Table 6) showed that the coefficient of variation (CV) values for intra-batch reproducibility tests ranged from 1.44% to 5.53%, and the CV values for inter-batch reproducibility tests ranged from 1.30% to 5.11%, both less than 6%, indicating good reproducibility of the kit.

[0156] Table 6 Kit repeatability test results

[0157]

[0158] 5.4 Shelf Life

[0159] The kits (Box A at 2-8°C, Box B at -20°C) were stored for 3, 6, 12, and 15 months for sensitivity, specificity, and repeatability testing. The test results were all consistent at 100%, indicating that the kits could be stored for up to 15 months.

[0160] 5.5 Clinical Application

[0161] 5.5.1 Semi-finished product inspection

[0162] According to the method of Example 1, 10 batches of CATHAY-type O virus-like particles, i.e., CATHAY-type O virus subunit vaccine semi-finished products, were prepared. The kit was used for detection and the results were compared with those of the BCA test. The results (see Table 7) showed that the relative deviations between the detection results of the kit and the BCA test were less than 10%, indicating that the kit was well applied in detecting semi-finished products.

[0163] Table 7 Semi-finished product test results

[0164]

[0165] 5.5.2 Finished product testing

[0166] The CATHAY-type O-type FMD virus-like particles, SEA-type O-type FMD virus-like particles, and A-type FMD virus-like particles prepared in Example 1 were emulsified with ISA 206 adjuvant according to the antigenic components and contents in Table 8 to prepare a finished product containing a CATHAY-type O-type FMD virus subunit vaccine. The vaccine was demulsified with n-pentanol, and the aqueous phase was extracted and tested using this kit. The results (see Table 8) show that this kit can specifically detect CATHAY-type O-type FMD virus in single-component, two-component, and three-component vaccines containing CATHAY-type O virus, with relative deviations from the theoretical value of less than 3%, demonstrating that this kit is suitable for detecting finished products.

[0167] Table 8 Finished product test results

[0168]

[0169] In summary, the kit prepared by the present invention overcomes the shortcomings of protein quantification methods such as BCA that cannot specifically detect proteins. The dual monoclonal antibody ELISA method is established using monoclonal antibody technology, which can conveniently, quickly, and specifically detect CATHAY type O foot-and-mouth disease virus in finished products and semi-finished products with good reproducibility and a sensitivity of up to 7.8 μg / ml, providing support for the quality control of foot-and-mouth disease virus subunit vaccines.

[0170] Example 6 Preparation and Application of Genetically Engineered Antibodies

[0171] The heavy chain variable region and light chain variable region gene sequences of the monoclonal antibodies 4B12 and 10G4 in Example 2 were amplified, respectively. The heavy chain variable region genes and light chain variable region genes were linked by connecting peptides to construct recombinant plasmids 4B12-ScFv, 10G4-ScFv, 4B12 heavy + 10G4 light-ScFv, and 10G4 heavy + 4B12 light-ScFv, respectively. The ScFv genes were inserted into the pCDNA-3.1 vector, and the pCDNA-4B12-ScFv, pCDNA-10G4-ScFv, pCDNA-4B12 heavy + 10G4 light-ScFv, and pCDNA-10G4 heavy + 4B12 light-ScFv eukaryotic expression systems were constructed, respectively, and MDCK cells were transfected for expression.

[0172] The CATHAY-type O foot-and-mouth disease virus-like particles prepared in Example 1 were used as coating antigens and coated on a microtiter plate at 0.2 μg / ml, 100 μl / well, followed by blocking with PBS containing 5% w / v skim milk. The expressed single-chain antibodies 4B12, 10G4, 4B12 heavy + 10G4 light, and 10G4 heavy + 4B12 light were titered, and the titers were 1:3200, 1:1600, 1:1600, and 1:3200, respectively, indicating that the single-chain antibodies had good reactivity with the CATHAY-type O foot-and-mouth disease virus-like particles.

[0173] The above results show that SEQ.ID No.1, SEQ.ID No.2, SEQ.ID No.3 and SEQ.ID No.4 can be used for the preparation of genetically engineered antibodies against CATHAY type O foot-and-mouth disease virus-like particles.

[0174] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention. SEQUENCE LISTING <110> Luoyang Putai Biotechnology Co., Ltd. <120> CATHAY type O foot-and-mouth disease virus monoclonal antibody and its preparation kit and application <160> 4 <170> PatentIn version 3.3 <210> 1 <211> 357 <212> DNA <213> Hybridoma <400> 1 cagatccagt tggtgcaatc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60 tcctgcaagg cttctgggta taccttcaca cactatggga tgaactggat gaagcaggct 120 ccaggagagg gtttaaagtg gatgggctgg ataaacacct acactggaga gctaacatat 180 gctgatgact tcaagggacg gtttgtcttc tctttggaaa cctctgccag cactgtctat 240 ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtac aagggtgcgc 300 gacgacgggg cctggtttgc ttactggggc caagggactc tggtcactgt ctctgca 357 <210> 2 <211> 306 <212> DNA <213> Hybridoma <400> 2 caaattgttc tcacccagtc accagcaatc atgagcgcaa caccagggga gagggtcaca 60 atgacctgca gtgccagctc aggtgtaagt tacatgtact ggtatcagca gaagccagga 120 tcatccccca gactcctgat atatgccaca tccaacttgg cttctggagt ccctgttcgc 180 tttagtggca gtggctctgg gacctcttac tctctcacaa tcagccgaat ggaggctgaa 240 gatgctgcaa cttattactg ccagcagtgg acttattacc cgttcacgtt cggtgggggg 300 accgag 306 <210> 3 <211> 357 <212> DNA <213> Hybridoma <400> 3 cagatccagt tggtgcaatc tgcgcctgag ctgaagaaac ctggagaaac agtcaagatc 60 tcgtgcaagg cttctggtta taccttcaca cactatggga tgaattggat gaagcaggct 120 ccaggagagg gtttaaaatg gatgggctgg ataaacacgt acactggaga gctaacatat 180 gccgatgact tcaagggacg gttcgtcttc tctttggaaa cctctgccag cactgtctat 240 ttgcagctta acaacctcaa gaatgaggac acggctacat atttttgtac aagggtgcgc 300 gacgacgccg cctggtttgc ttactggggc caggggactc tggtcactgt ctctgca 357 <210> 4 <211> 336 <212> DNA <213> Hybridoma cell <400> 4 gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctaggaca gagggccacc 60 atatcctgcc aagccagcga aagtgtcagt tttgctggta caagtttaat gcactggtac 120 caacagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct 180 ggagtccctg ccaggttcag tggcagtggg tctgagtcag acttcactct caccatcgat 240 cctgtggagg aagatgatgc tgcaatgtat tactgtatgc aaagtatgga agatccgtac 300 acgttcggag gggggaccaa gctggaaata aaacgg 336

Claims

1. An antibody or antigen-binding fragment that specifically binds to a CATHAY-type O foot-and-mouth disease virus antigen, wherein: The nucleotide sequence of the heavy chain variable region of the antibody or antigen-binding fragment is SEQ.ID No.1, and the nucleotide sequence of the light chain variable region of the antibody or antigen-binding fragment is SEQ.ID No.2; the antibody or the antigen-binding fragment specifically binds to CATHAY type O foot-and-mouth disease virus-like particles.

2. The antibody or antigen-binding fragment according to claim 1, wherein The antibody is a monoclonal antibody or a genetically engineered antibody.

3. The antibody or antigen-binding fragment according to claim 2, wherein The genetically engineered antibodies include single-chain antibodies, chimeric monoclonal antibodies, and reshaped monoclonal antibodies.

4. The antibody or antigen-binding fragment according to claim 1, wherein The antibody is the monoclonal antibody 4B12, the nucleotide sequence of the heavy chain variable region of the monoclonal antibody 4B12 is SEQ.ID No.1, and the nucleotide sequence of the light chain variable region of the monoclonal antibody 4B12 is SEQ.ID No.

2.

5. An antibody or antigen-binding fragment that specifically binds to a CATHAY-type O foot-and-mouth disease virus antigen, wherein: The nucleotide sequence of the heavy chain variable region of the antibody or antigen-binding fragment is SEQ.ID No.3, and the nucleotide sequence of the light chain variable region of the antibody or antigen-binding fragment is SEQ.ID No.4; the antibody or the antigen-binding fragment specifically binds to CATHAY type O foot-and-mouth disease virus-like particles.

6. The antibody or antigen-binding fragment according to claim 5, wherein The antibody is a monoclonal antibody or a genetically engineered antibody.

7. The antibody or antigen-binding fragment according to claim 6, wherein The genetically engineered antibodies include single-chain antibodies, chimeric monoclonal antibodies, and reshaped monoclonal antibodies.

8. The antibody or antigen-binding fragment according to claim 5, wherein The antibody is the monoclonal antibody 10G4, the nucleotide sequence of the heavy chain variable region of the monoclonal antibody 10G4 is SEQ.ID No.3, and the nucleotide sequence of the light chain variable region of the monoclonal antibody 10G4 is SEQ.ID No.

4.

9. A kit for detecting CATHAY type O foot-and-mouth disease virus antigen, wherein: The kit comprises: a support medium coated with the monoclonal antibody 4B12 according to claim 4, the support medium being blocked with a blocking solution after being coated with the monoclonal antibody 4B12, and an enzyme-labeled monoclonal antibody 10G4 according to claim 8; 20× concentrated washing solution, wherein the 20× concentrated washing solution is a 0.1 M PBS solution with a pH of 7.4 containing 10% v / v Tween 20; Standard, the standard is CATHAY type O foot-and-mouth disease virus-like particles; A color developing solution, comprising color developing solution A and color developing solution B, wherein color developing solution A is an aqueous solution containing 14.7 g / L disodium hydrogen phosphate, 9.3 g / L citric acid, and 0.3 g / L urea peroxide, and color developing solution B is an aqueous solution containing 0.2 g / L tetramethylbenzenediamine (TMB) and 10% v / v anhydrous ethanol; The stop solution is 2M H2SO4 solution.

10. The kit according to claim 9, wherein The support medium coated with the monoclonal antibody 4B12 is a microtiter plate, the working concentration of the monoclonal antibody 4B12 is 0.05 μg / well to 0.1 μg / well, the titer of the enzyme-labeled monoclonal antibody 10G4 is 1:40 to 1:80, and the enzyme is horseradish peroxidase; The blocking solution is 0.1 M PBS with a pH of 7.4 containing 1% w / v soy protein, 1% w / v sucrose, and 0.05% v / v Tween 20; The standard substance is 1 mg / ml of CATHAY type O foot-and-mouth disease virus-like particles; The kit further includes a sample diluent, which is a 0.1 M PBS solution with a pH of 7.4 and containing 0.5% to 2% w / v sucrose, 1% w / v EDTA, and 0.004% w / v AW dye.

11. The kit according to claim 10, wherein The working concentration of the monoclonal antibody 4B12 was 0.1 μg / well, and the titer of the enzyme-labeled monoclonal antibody 10G4 was 1:

40.

12. A method for preparing the kit according to claim 9 or 10, wherein: The method comprises: Step (1) diluting the monoclonal antibody 4B12 with a carbonate buffer solution at pH 9.6 and coating the solution on a microtiter plate; Step (2) blocking the microtiter plate coated with the monoclonal antibody 4B12 in step (1) with the blocking solution; Step (3) preparing the sample diluent, the enzyme-labeled monoclonal antibody 10G4, the 20× concentrated washing solution, the standard, the color developing solution, and the stop solution; Step (4) assembles the closed microtiter plate of step (2), the sample diluent of step (3), the 20× concentrated washing solution, the color developing solution, and the stop solution into a kit A box, and assembles the standard of step (3) into a kit B box.

13. Use of the kit according to claim 9 or 10 in detecting the content of CATHAY-type O-type foot-and-mouth disease virus-like particles; wherein, The content of the CATHAY-type O-type foot-and-mouth disease virus-like particles is the content of the CATHAY-type O-type foot-and-mouth disease virus-like particles in a single vaccine, a multivalent vaccine, or a combined vaccine.

14. The use according to claim 13, wherein: The single vaccine, multivalent vaccine, or combination vaccine is a semi-finished vaccine or a finished vaccine.

15. A single-chain antibody, wherein The nucleotide sequence of the heavy chain variable region of the single-chain antibody is SEQ.ID No.1, or the nucleotide sequence of the heavy chain variable region of the single-chain antibody is SEQ.ID No.3; and the nucleotide sequence of the light chain variable region of the single-chain antibody is SEQ.ID No.2, or the nucleotide sequence of the light chain variable region of the single-chain antibody is SEQ.ID No.4, and the single-chain antibody specifically binds to CATHAY type O foot-and-mouth disease virus-like particles.

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