Recombinant antibody, kit containing recombinant antibody and application of recombinant antibody to diagnosis of African swine fever virus

Abstract

The present disclosure relates to a recombinant antibody, or a fragment thereof, for use in the detection of African swine fever virus (ASFV). The present disclosure also provides a kit comprising the recombinant antibody, and a method of diagnosing ASFV infection using the recombinant antibody or the kit.

Description

Background of the Invention 1. Technical Field

[0002] The present disclosure relates to the field of disease diagnosis. Specifically, the present disclosure relates to recombinant antibodies specific for African swine fever virus (ASFV), and their use in diagnosing ASFV infection. 2. Background technology

[0004] ASFV is a large, enveloped, double-stranded DNA virus belonging to the genus Asfivirus and the only species in the family Asfarviridae. ASFV is the causative agent of African swine fever (ASF), a highly contagious viral disease of domestic pigs that causes severe bleeding in infected individuals. ASF first broke out in Kenya in 1914. Since 2007, the disease has continued to spread throughout Europe and has been reported in China, the Philippines, Vietnam, South Korea, and East Timor in 2019. ASF causes high economic losses in many affected countries and has been listed as the highest level disease of domestic pigs by the World Organization for Animal Health. To date, there is still no vaccine to control ASF. The only means of control are early detection of cases, culling, and strict biosecurity measures to prevent the spread of the disease.

[0005] In view of this, the relevant field is in urgent need of a reagent and / or method for early detection of ASFV infection so as to prevent and control the spread of the disease in a timely manner. Summary of the invention

[0006] A brief summary of the disclosure is presented below to facilitate a basic understanding of the disclosure by the reader. This summary is not an extensive overview of the disclosure, nor is it intended to identify the key / determining components of the disclosure or to outline the scope of the invention. Its sole purpose is to present certain concepts of the disclosure in a simplified form as a prelude to a more detailed description that will be presented later.

[0007] As embodied and broadly described herein, one aspect of the present disclosure relates to a recombinant antibody or fragment thereof. According to an embodiment of the present disclosure, the recombinant antibody or antibody fragment comprises a light chain variable (VL) domain and a heavy chain variable (VH) domain, wherein the VL domain comprises a first light chain complementarity determining region (CDR-L1), a second light chain complementarity determining region (CDR-L2), and a third light chain complementarity determining region (CDR-L3); and the VH domain comprises a first heavy chain complementarity determining region (CDR-H1), a second heavy chain complementarity determining region (CDR-H2), and a third heavy chain complementarity determining region (CDR-H3).

[0008] According to certain embodiments of the present disclosure, the CDR-L1, CDR-L2 and CDR-L3 have the amino acid sequences of sequence numbers: 1-3, respectively, and the CDR-H1, CDR-H2 and CDR-H3 have the amino acid sequences of sequence numbers: 4-6, respectively. According to certain preferred embodiments, the VL domain comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 37, and the VH domain comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 38. In certain operative embodiments, the VL domain and VH domain of the recombinant antibody or antibody fragment respectively comprise an amino acid sequence having 100% sequence similarity to sequence numbers: 37 and 38.

[0009] According to certain embodiments of the present disclosure, the CDR-L1, CDR-L2 and CDR-L3 have amino acid sequences of sequence numbers: 7-9, respectively, and the CDR-H1, CDR-H2 and CDR-H3 have amino acid sequences of sequence numbers: 10-12, respectively. According to certain preferred embodiments, the VL domain comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 39, and the VH domain comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 40. In certain operative embodiments, the VL domain and VH domain of the recombinant antibody or antibody fragment respectively comprise an amino acid sequence having 100% sequence similarity to sequence numbers: 39 and 40.

[0010] According to certain embodiments of the present disclosure, the CDR-L1, CDR-L2 and CDR-L3 have amino acid sequences of sequence numbers: 13-15, respectively, and the CDR-H1, CDR-H2 and CDR-H3 have amino acid sequences of sequence numbers: 16-18, respectively. According to certain preferred embodiments, the VL domain comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 41, and the VH domain comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 42. In certain embodiments, the VL domain and VH domain of the recombinant antibody or antibody fragment respectively comprise an amino acid sequence having 100% sequence similarity to sequence numbers: 41 and 42.

[0011] According to certain embodiments of the present disclosure, the CDR-L1, CDR-L2 and CDR-L3 have amino acid sequences of sequence numbers: 19-21, respectively, and the CDR-H1, CDR-H2 and CDR-H3 have amino acid sequences of sequence numbers: 22-24, respectively. According to a preferred embodiment, the VL domain comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 43, and the VH domain comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 44. In certain embodiments, the VL domain and VH domain of the recombinant antibody or antibody fragment respectively comprise an amino acid sequence having 100% sequence similarity to sequence numbers: 43 and 44.

[0012] According to certain embodiments of the present disclosure, the CDR-L1, CDR-L2 and CDR-L3 have amino acid sequences of sequence numbers: 25-27, respectively, and the CDR-H1, CDR-H2 and CDR-H3 have amino acid sequences of sequence numbers: 28-30, respectively. According to certain preferred embodiments, the VL domain comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 45, and the VH domain comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 46. In certain operative embodiments, the VL domain and VH domain of the recombinant antibody or antibody fragment respectively comprise an amino acid sequence having 100% sequence similarity to sequence numbers: 45 and 46.

[0013] According to certain embodiments of the present disclosure, the CDR-L1, CDR-L2 and CDR-L3 have amino acid sequences of sequence numbers: 31-33, respectively, and the CDR-H1, CDR-H2 and CDR-H3 have amino acid sequences of sequence numbers: 34-36, respectively. According to certain preferred embodiments, the VL domain comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 47, and the VH domain comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 48. In certain specific embodiments, the VL domain and VH domain of the recombinant antibody or antibody fragment respectively comprise an amino acid sequence having 100% sequence similarity to sequence numbers: 47 and 48.

[0014] Another aspect of the present disclosure is a kit for detecting the presence of ASFV in a biological sample. The kit comprises a first recombinant antibody, a second recombinant antibody, and a container containing the first and second recombinant antibodies, wherein the first and second recombinant antibodies are individually selected from the recombinant antibodies described in the first aspect of the present disclosure. According to certain embodiments, one of the first and second recombinant antibodies is used as a capture antibody in a detection technique, and the other is used as a detection antibody in a detection technique; exemplary detection techniques include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay (LFIA), and western blotting (WB).

[0015] According to certain embodiments, CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 of the first recombinant antibody respectively comprise the amino acid sequences of sequence numbers: 1-6, and CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 of the second recombinant antibody respectively comprise the amino acid sequences of sequence numbers: 31-36. In certain exemplary embodiments, the VL domain and VH domain of the first recombinant antibody respectively comprise amino acid sequences having at least 85% sequence similarity to sequence numbers: 37 and 38, and the VL domain and VH domain of the second recombinant antibody respectively comprise amino acid sequences having at least 85% sequence similarity to sequence numbers: 47 and 48. In a specific embodiment, the VL domain and VH domain of the first recombinant antibody respectively comprise an amino acid sequence having 100% sequence similarity to sequence numbers: 37 and 38, and the VL domain and VH domain of the second recombinant antibody respectively comprise an amino acid sequence having 100% sequence similarity to sequence numbers: 47 and 48.

[0016] According to certain embodiments, CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 of the first recombinant antibody respectively comprise the amino acid sequences of sequence numbers: 7-12, and CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 of the second recombinant antibody respectively comprise the amino acid sequences of sequence numbers: 25-30. In certain exemplary embodiments, the VL domain and VH domain of the first recombinant antibody respectively comprise amino acid sequences having at least 85% sequence similarity to sequence numbers: 39 and 40, and the VL domain and VH domain of the second recombinant antibody respectively comprise amino acid sequences having at least 85% sequence similarity to sequence numbers: 45 and 46. In a specific embodiment, the VL domain and VH domain of the first recombinant antibody respectively comprise an amino acid sequence having 100% sequence similarity to sequence numbers: 39 and 40, and the VL domain and VH domain of the second recombinant antibody respectively comprise an amino acid sequence having 100% sequence similarity to sequence numbers: 45 and 46.

[0017] According to alternative embodiments, the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 of the first recombinant antibody respectively comprise the amino acid sequences of sequence numbers: 13-18, and the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 of the second recombinant antibody respectively comprise the amino acid sequences of sequence numbers: 19-24. In certain exemplary embodiments, the VL domain and VH domain of the first recombinant antibody respectively comprise amino acid sequences having at least 85% sequence similarity to sequence numbers: 41 and 42, and the VL domain and VH domain of the second recombinant antibody respectively comprise amino acid sequences having at least 85% sequence similarity to sequence numbers: 43 and 44. In a specific embodiment, the VL domain and VH domain of the first recombinant antibody respectively comprise amino acid sequences having 100% sequence similarity to sequence numbers: 41 and 42, and the VL domain and VH domain of the second recombinant antibody respectively comprise amino acid sequences having 100% sequence similarity to sequence numbers: 43 and 44.

[0018] The present disclosure also provides a method for confirming whether an individual is infected with ASFV by using a biological sample isolated from an individual. The method comprises detecting the presence or absence of ASFV viral protein in the biological sample by using an antibody fragment, a recombinant antibody or a kit of the present disclosure, wherein the presence of the viral protein indicates that the individual is infected with ASFV.

[0019] According to certain embodiments, the viral protein is a 30-kDa phosphoprotein (p30 protein).

[0020] Based on the above results, a person with ordinary knowledge in the field of the present invention or a clinical practitioner can timely administer appropriate treatment to an individual in need (e.g., pigs infected with ASFV). Specifically, if a viral protein (e.g., p30 protein) is present in a biological sample of an individual, an effective amount of anti-viral treatment (e.g., apigenin, enrofloxacin, grepafloxacin, balofloxacin, tosufloxacin, gatifloxacin, garenoxacin, or a combination thereof) can be administered to the individual to reduce and / or improve the symptoms associated with ASFV infection. Alternatively, the ASFV-infected pigs can be separated from other livestock on the farm to prevent and control the spread of ASFV.

[0021] After referring to the following detailed description and the accompanying drawings, many of the accompanying features and advantages of the present disclosure will be easily understood. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present disclosure will become more apparent after referring to the following detailed description and the accompanying drawings, in which:

[0023] Figures 1A to 1C The photographs shown in Example 2 of the present disclosure respectively illustrate the binding affinity and specificity of specific antibodies to the p30 protein of ASFV, wherein the p30 protein is expressed by HEK293 cells. Figure 1A : Binding affinity (panel (A)) and binding specificity (panel (B)) of the D96C77 antibody pair to p30 protein. Figure 1B : Binding affinity (panel (A)) and binding specificity (panel (B)) of D91C90 antibody to p30 protein. Figure 1C : Binding affinity (small picture (A)) and binding specificity (small picture (B)) of D97C52 antibody to p30 protein. Figure 1B and 1C In the panel (A), the detection limit is indicated by the symbol "*". The D96C77 antibody pair includes p30-077 IgG as a capture antibody and p30-096 IgG as a detection antibody; the D91C90 antibody pair includes p30-090 IgG as a capture antibody and p30-091 IgG as a detection antibody; and the D97C52 antibody pair includes p30-052 IgG as a capture antibody and p30-097 IgG as a detection antibody.

[0024] Figures 2A to 2CThe photographs shown in Example 2 of the present disclosure respectively illustrate the binding affinity and specificity of specific antibodies to the p30 protein of ASFV, wherein the p30 protein is expressed by Escherichia coli (E. coli). Figure 2A : Binding affinity (panel (A)) and binding specificity (panel (B)) of D96C77 antibody to p30 protein. Figure 2B : Binding affinity (panel (A)) and binding specificity (panel (B)) of D91C90 antibody to p30 protein. Figure 2C : Binding affinity (small picture (A)) and binding specificity (small picture (B)) of D97C52 antibody to p30 protein. Figure 2B and 2C In the panel (A), the detection limit is indicated by the symbol “*”. DETAILED DESCRIPTION

[0025] In order to make the description of the present disclosure more detailed and complete, the following is an illustrative description of the implementation and specific embodiments of the present invention; however, this is not the only form of implementing or using the specific embodiments of the present invention. The implementation covers the features of multiple specific embodiments and the method steps and sequences used to construct and operate these specific embodiments. However, other specific embodiments can also be used to achieve the same or equal functions and step sequences.

[0026] I. Definitions

[0027] For convenience, specific terms used in this specification, embodiments and the attached claims are collected here. Unless otherwise defined in the title of this book, the meaning of scientific and technical terms used herein is the same as that understood and used by those with ordinary knowledge in the technical field to which the present invention belongs. In addition, singular nouns used in this specification include plural forms of the nouns, and plural nouns used also include singular forms of the nouns, unless they conflict with the context. Specifically, in this specification and the claims, the singular form "a" (a and an) includes plural reference values, except where the context clearly indicates otherwise. In addition, in this specification and the claims, the expressions "at least one" and "one or more" have the same meaning, both of which represent one, two, three or more.

[0028] Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate values, the relevant numerical values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations caused by individual testing methods. In addition, the term "about" here generally refers to the actual value within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range. Alternatively, the term "about" means that the actual value falls within the acceptable standard error of the mean value, depending on the consideration of a person with ordinary knowledge in the technical field to which the present invention belongs. Except for the examples, or unless otherwise explicitly stated, it should be understood that all ranges, quantities, values ​​and percentages used herein (for example, to describe the amount of material, the length of time, temperature, operating conditions, quantitative ratios and the like) are modified by "about". Accordingly, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the scope of the patent application are all approximate values ​​and can be changed as needed. At least, these numerical parameters should be understood as the number of significant digits indicated and the values ​​obtained by applying the general rounding method.

[0029] The term "antibody" has the broadest meaning in the present disclosure, and specifically includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multi-specific or multivalent antibodies (e.g., bi-specific antibodies), and antibody fragments having the desired biological activity. The term "antibody fragment" (antibody fragment or the fragment of an antibody) refers to a portion of a full-length antibody, generally an antigen-binding domain or a variable domain (i.e., a VL domain and a VH domain) of a full-length antibody. Exemplary antibody fragments include antigen-binding fragment (Fab), Fab', F(ab')2, single-chain variable fragment (scFv), diabody, linear antibody, single-chain antibody molecule, and multifunctional antibodies formed by antibody fragments.

[0030] The term "complementarity determining region" (CDR) refers to the hypervariable region of an antibody molecule that forms a surface complementary to the 3-dimensional surface of the bound antigen. From N-terminus to C-terminus, each antibody's heavy and light chains contain three CDRs (i.e., CDR-1, CDR-2, and CDR-3). Therefore, a human leukocyte antigen-DR (HLA-DR) binding site includes a total of six CDRs, including three CDRs from the heavy chain variable domain (i.e., CDR-H1, CDR-H2, and CDR-H3), and three CDRs from the light chain variable domain (i.e., CDR-L1, CDR-L2, and CDR-L3). The amino acid residues of the CDRs are in close contact with the bound antigen, and the one that is most closely in contact with the antigen is usually the heavy chain CDR3.

[0031] As used herein, the term "variable region" of an antibody refers to the amino-terminal region of an antibody heavy or light chain. These regions are generally the most variable parts of an antibody and contain the antigen binding site. The term "variable" refers to the fact that some parts of the variable region differ greatly in sequence between different antibodies and are used to determine the binding and specificity of each specific antibody to its specific antigen. However, this variability is not evenly distributed within the variable region of an antibody. It is concentrated in three segments of the complementarity determining regions or hypervariable regions of the light and heavy chains. The highly conserved parts of the variable region are called frameworks (FRs). The variable region of natural heavy and light chains contains four FR regions, which mainly adopt a β-sheet configuration and are connected by three CDRs, which form a loop connecting the β-sheet structure and in some cases form part of the β-sheet structure. The CDRs in each chain are tightly connected by the FR region and together with the CDRs from the other chain form the antigen binding site of the antibody. The constant region is not directly involved in the binding of an antibody to an antigen, but exhibits various effector functions, for example, participating in the antibody-dependent cellular toxicity effect of the antibody.

[0032] "Percentage (%) sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular reference sequence in any of the amino acid sequences disclosed herein. Gaps may be introduced as needed when aligning the sequences to achieve the maximum percentage of sequence identity, and any conservative substitutions are not considered as part of the sequence identity. Alignment can be performed using various methods known to those skilled in the art to determine the percentage of sequence identity. For example, the alignment can be performed using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters for measuring alignment, including any desired algorithm to maximize alignment values ​​for the full length of the aligned sequences. For the purposes of this article, the sequence alignment between two amino acid sequences is analyzed using the computer program Blastp (protein-protein BLAST) provided by the National Center for Biotechnology Information (NCBI). The sequence similarity percentage between a specific sequence A and a specific sequence B (which can also be expressed as a specific percentage of sequence similarity between a specific sequence A and a specific sequence B) is calculated using the following formula: Where X is the number of amino acid residues that are evaluated as identical matches in the alignment of A and B in the program after the sequence alignment program BLAST, and Y is the total number of amino acid residues in the specific sequence B.

[0033] As discussed herein, antibodies with slight variations in their amino acid sequences are considered to be encompassed by the present disclosure and the claimed inventive concepts, as long as the variability of the amino acid sequence maintains at least 85% sequence similarity, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence similarity. The antibodies of the present disclosure may be specifically modified to alter the characteristics of the peptide that are unrelated to its physiological activity. For example, certain amino acids may be altered and / or deleted without affecting the physiological activity of the antibodies of the present invention (i.e., the ability to bind to African swine fever virus). In particular, conservative amino acid substitutions are contemplated. Conservative replacements refer to substitutions that occur in a family of amino acids whose side chains are related. In general, the amino acids encoded by genes can be divided into four major categories: (1) acidic amino acids, namely aspartate and glutamate; (2) basic amino acids, namely lysine, arginine, and histidine; (3) non-polar amino acids, namely alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids, namely glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. A preferred classification is that serine and threonine are aliphatic-hydroxy; asparagine and glutamine are amide-containing; alanine, valine, leucine, and isoleucine are aliphatic; and phenylalanine, tryptophan, and tyrosine are aromatic. For example, it is reasonable to expect that a single substitution of leucine for isoleucine or valine, aspartic acid for glutamic acid, threonine for serine, or a similar substitution of an amino acid for a structurally related amino acid will not significantly affect the binding or properties of the resulting molecule, especially when the substitution does not involve an amino acid within a framework position. By measuring the specific activity of the peptide derivative, it can be easily confirmed whether the amino acid change will produce a functional peptide.Those skilled in the art can easily prepare fragments or analogs of antibodies. Preferably, the amino and carboxyl termini of the fragments or analogs are located near the boundaries of the functional regions.

[0034] Unless otherwise specified, the term "subject" refers to both male and female individuals.

[0035] II. Description of the Invention

[0036] (II-1) Method for screening antibody fragments that specifically bind to ASFV

[0037] The first aspect of the present disclosure is a method for screening antibody fragments that can specifically bind to ASFV. According to an embodiment of the present disclosure, the method comprises the following steps: (a) providing a phage-expressed single-chain variable fragment (scFv) antibody library, comprising a plurality of scFvs expressed by phage, wherein the VH domain of each scFv expressed by the phage has a binding affinity to protein A, and the VL domain of each scFv expressed by the phage has a binding affinity to protein L; (b) exposing the scFv antibody library expressed by phage in step (a) to a viral protein derived from ASFV; (c) screening a plurality of phages from the scFv antibody library expressed by the phages in step (b), each of which expresses an scFv having binding affinity to the viral protein; (d) allowing the plurality of phages screened in step (c) to express a plurality of soluble scFvs respectively; (e) exposing the plurality of soluble scFvs of step (d) to the viral protein; (f) respectively confirming the binding affinity of the plurality of soluble scFvs in step (e) to the viral protein; and (g) Based on the result determined in step (f), a soluble scFv having a binding affinity to the viral protein better than other soluble scFvs is selected from the plurality of soluble scFvs as an antibody fragment.

[0038] In step (a), a scFv antibody library expressed by phage is provided. According to an embodiment of the present disclosure, the framework of the scFv antibody library expressed by phage is based on the human IGKV1-NL1*01 / IGHV3-23*04 germ cell sequence, and its CDR sequence is diversified by PCR reaction using specific primers, including CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 sequences. After screening with protein A and protein L, a scFv antibody library expressed by phage (hereinafter referred to as "GH2 antibody library") can be prepared, wherein each scFv in the plurality of scFv antibody libraries expressed by phage has a VH domain that can bind to protein A, and a VL domain that can bind to protein L. The scFv antibody libraries expressed by phage can be constructed using the methods described in the PCT applications PCT / US2016 / 19128 and PCT / US2018 / 56627, and the public documents of Ing-Chien Chen et al. (Highthroughput discovery of influenza virus neutralizing antibodies from phage-displayed synthetic antibody libraries, Scientific Reports 7, Article number: 14455 (2017)). The application and the public document are hereby incorporated by reference in their entirety.

[0039] In step (b), the GH2 antibody library is exposed to a viral protein derived from ASFV. According to certain embodiments, the viral protein is p30 protein, which is an early protein that is part of the inner membrane of ASFV particles. In certain operative embodiments, the p30 protein of ASFV comprises the amino acid sequence of SEQ ID NO: 49. According to certain operative embodiments, the p30 protein is immobilized on a matrix (e.g., agarose resin or polyacrylamide) and then mixed with the GH2 antibody library of the present disclosure.

[0040] In step (c), a plurality of phages are selected from the GH2 antibody library, wherein the selected phages express scFvs having binding affinity to the viral protein (i.e., p30 protein). Specifically, the product of step (b) is treated with an extraction buffer, which is usually an acidic solution (e.g., a glycine solution, pH 2.2), to destroy the binding between the viral protein and the scFv expressed by the phage. In this way, a plurality of phages expressing scFvs having binding affinity to the viral protein are collected.

[0041] Optionally, step (c) is performed under acidic conditions. Specifically, the product of step (b) can be treated with acid (e.g., using a washing buffer having a pH value between 5 and 7, for example, using a washing buffer having a pH value of 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7; preferably, using a washing buffer having a pH value of 5.0), followed by the aforementioned elution step to collect the plurality of phages.

[0042] Next, in step (d), the plurality of phages screened in step (c) are placed under appropriate conditions so that they can produce a plurality of soluble scFvs. This step can be performed using methods known to those skilled in the art. According to certain embodiments of the present disclosure, the lac operon can be used to drive the expression of the VH domain and the VL domain; as is well known to those skilled in the art, the lac operon can be triggered by isopropyl-thio-β-D-galactoside (IPTG), thereby driving the expression of downstream genes (i.e., genes encoding the VH domain and the VL domain). The scFv produced thereby is then secreted into the supernatant of the culture medium and can be collected therefrom.

[0043] In step (e), the soluble scFv produced in step (d) is mixed with viral protein (ie, p31 protein) to form a protein-scFv complex.

[0044] Next, in step (f), the amount of the protein-scFv complex formed in step (e) can be confirmed by a method known to those skilled in the art for analyzing the binding affinity between two molecules (e.g., the binding affinity of an antibody to an antigen); for example, ELISA, Western blot, flow cytometry, surface plasmon resonance (SPR), or LFIA can be used for detection. Generally speaking, the amount of the protein-scFv complex is proportional to the binding affinity between the scFv and the viral protein (i.e., p30 protein). According to an operational embodiment, the amount of the protein-scFv complex (i.e., the binding affinity between the soluble scFv and the viral protein) is confirmed by ELISA.

[0045] Finally, in step (g), an antibody fragment is selected based on the binding affinity determined in step (f). More specifically, a soluble scFv is selected as an antibody fragment from a plurality of soluble scFvs, wherein the soluble scFv exhibits a binding affinity for the viral protein (i.e., p30 protein) that is superior to other soluble scFvs.

[0046] According to certain embodiments of the present disclosure, the six antibody fragments screened out through different screening procedures are named "p30-052 scFv", "p30-077 scFv", "p30-090 scFv", "p30-091 scFv", "p30-096scFv" and "p30-097 scFv", respectively.

[0047] (II-2) Method for preparing recombinant antibodies

[0048] The antibody fragments screened in part (II-1) of the present disclosure can be used to prepare a recombinant antibody, wherein the recombinant antibody structurally comprises a VL domain, a light chain constant (CL) domain, a VH domain, and a heavy chain constant (CH) domain. The method for preparing a recombinant antibody using the screened antibody fragments comprises the following steps: (1) Providing a phage expressing a specific antibody fragment; (2) extracting phagemid DNA corresponding to the phage of step (a); (3) using the phagosome DNA of step (2) as a template, and amplifying by PCR a first nucleic acid sequence encoding the VH domain and a second nucleic acid sequence encoding the VL domain; (4) inserting the first and second nucleic acid sequences into an expression vector comprising a third and a fourth nucleic acid sequence, wherein the third nucleic acid sequence is used to encode a CH domain of an immunoglobulin, and the fourth nucleic acid sequence is used to encode a CL domain of the immunoglobulin; and (5) Transfecting the expression vector of step (4) (comprising the first, second, third and fourth nucleic acid sequences) into a host cell to produce the recombinant antibody.

[0049] In the method of the present disclosure, the phage expressing the antibody fragment is used as the starting material for preparing the recombinant antibody (ie, step (1)).

[0050] Next, as described in step (2), the phage DNA corresponding to the phage expressing the antibody fragment is extracted. Depending on the desired purpose, the phage can be extracted by lysing the phage; or, the phage can be obtained from a bacterial plant (i.e., a bacterial strain containing the phage). The purpose of extracting phage DNA from phage or bacterial plants can be achieved by any conventional DNA extraction technique; for example, the phenol / chloroform method and a surfactant (e.g., sodium dodecyl sulfate, NP-40 and X-100) / acetic acid method for extraction.

[0051] In step (3), the extracted phagosome DNA is used as a template to amplify the first nucleic acid sequence encoding CDR-H1, CDR-H2 and CDR-H3 (wherein specific primers (forward primer: sequence number: 50; reverse primer: sequence number: 51) are used for PCR); and the second nucleic acid sequence encoding CDR-L1, CDR-L2 and CDR-L3 (wherein specific primers (forward primer: sequence number: 52; reverse primer: sequence number: 53) are used for PCR).

[0052] In step (4), the amplified first and second nucleic acid sequences are inserted into an expression vector, wherein the expression vector comprises a third nucleic acid sequence for encoding a heavy chain constant domain of an immunoglobulin, and a fourth nucleic acid sequence for encoding a light chain constant domain of the immunoglobulin. It is understood that the immunoglobulin can be any one of IgG, IgA, IgD, IgE and IgM. In a preferred embodiment of the present disclosure, the immunoglobulin is IgG. Specifically, the first and second nucleic acid sequences are first connected by a linker, wherein the linker is amplified from a plgG vector using PCR. According to an embodiment of the present disclosure, the linker sequentially comprises: a CL domain, a bovine growth hormone (BGH), a polyadenylation (polyA) signal, a human CMV promoter, and an IgG heavy chain signal peptide. For the complementary sequence between the 3'-end of the second nucleic acid sequence and the 5'-end of the linker, the complementary sequence between the 3'-end of the linker and the 5'-end of the first nucleic acid sequence, the second nucleic acid sequence, the linker and the first nucleic acid sequence can be assembled sequentially by overlap extension polymerase chain reaction (OE-PCR). Then, the assembly product is inserted into the pIgG expression vector by using restriction enzymes. Structurally, the constructed expression vector sequentially comprises: a first human CMV promoter, a signal peptide of an IgG light chain, a second nucleic acid sequence, a CL domain, a first BGH-polyA signal, a second human CMV promoter, a signal peptide of an IgG heavy chain, a first nucleic acid sequence, a CH domain and a second BGH-polyA signal, wherein the second nucleic acid sequence and the CL domain are driven by the first human CMV promoter to express the light chain of the recombinant antibody, and the first nucleic acid sequence and the CH domain are driven by the second human CMV promoter to express the heavy chain of the recombinant antibody.

[0053] In step (5), the expression vector constructed in step (4) is transfected into a host cell to produce the recombinant antibody of the present disclosure. The host cell commonly used is a mammalian cell, such as HEK293 cells. Transfection can be performed by methods well known to those skilled in the art, including chemical-based methods (e.g., calcium phosphate, liposomes, and cationic polymers), non-chemical methods (e.g., electroporation, cells queezing, sonoporation, optical transfection, protoplast fusion, and hydrodynamic delivery), particle-based methods (e.g., gene gun, magnetofection, and impalefection), and methods using viruses (e.g., adenoviral vectors, sindbis viral vectors, and lentiviral vectors). The recombinant antibodies thus prepared are secreted into the supernatant of the culture medium and can be purified therefrom by any purification method known to those skilled in the art; for example, purification can be achieved by affinity binding with protein A or protein G.

[0054] According to certain embodiments of the present disclosure, six recombinant antibodies prepared using the antibody fragments of part (II-1) of the present disclosure are named "p30-052 IgG", "p30-077 IgG", "p30-090 IgG", "p30-091IgG", "p30-096 IgG" and "p30-097 IgG", respectively.

[0055] (II-3) Recombinant Antibodies or Fragments thereof

[0056] According to certain embodiments, the antibody fragments described in part (II-1) of the present disclosure (including p30-052, p30-077, p30-090, p30-091, p30-096 and p30-097 scFv) and the recombinant antibodies prepared by part (II-2) of the present disclosure (including p30-052, p30-077, p30-090, p30-091, p30-096 and p30-097 IgG) structurally comprise a VL domain and a VH domain, wherein the VL domain comprises CDR-L1, CDR-L2 and CDR-L3, and the VH domain comprises CDR-H1, CDR-H2 and CDR-H3.

[0057] According to certain embodiments, CDR-L1, CDR-L2 and CDR-L3 of p30-052 scFv or p30-052 IgG respectively have the amino acid sequences of sequence numbers: 1-3 (i.e., they respectively have amino acid sequences with 100% sequence similarity to sequence numbers: 1-3), and CDR-H1, CDR-H2 and CDR-H3 of p30-052 scFv or p30-052 IgG respectively have the amino acid sequences of sequence numbers: 4-6 (i.e., they respectively have amino acid sequences with 100% sequence similarity to sequence numbers: 4-6). According to certain embodiments, the VL domain of p30-052 scFv or p30-052 IgG comprises an amino acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence similarity to SEQ ID NO: 37; and the VH domain of p30-052 scFv or p30-052 IgG comprises an amino acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence similarity to SEQ ID NO: 38. It is understood that the sequences of the VL domain and the VH domain (e.g., framework sequences) may differ (e.g., by substitution of conservative or non-conservative amino acid residues) without affecting the binding affinity and / or specificity of the antibodies of the present disclosure. Preferably, the sequences of the VL domain and the VH domain are conservatively substituted by one or more appropriate amino acids with similar properties; for example, leucine (a non-polar amino acid residue) is substituted with isoleucine, alanine, valine, proline, phenylalanine or tryptophan (another non-polar amino acid residue); aspartic acid (an acidic amino acid residue) is substituted with glutamic acid (another acidic amino acid residue); or lysine (a basic amino acid residue) is substituted with arginine or histidine (another basic amino acid residue). According to a preferred embodiment, the VL domain and the VH domain of p30-052 scFv or p30-052 IgG respectively comprise amino acid sequences having at least 90% sequence similarity to SEQ ID NOs: 37 and 38. More preferably, the VL domain and VH domain of p30-052 scFv or p30-052 IgG comprise amino acid sequences having at least 95% sequence similarity to SEQ ID NOs: 37 and 38, respectively.In an operational embodiment of the present disclosure, the VL domain of p30-052 scFv or p30-052 IgG has an amino acid sequence of sequence number: 37 (i.e., an amino acid sequence having 100% sequence similarity to sequence number: 37), and the VH domain of p30-052 scFv or p30-052 IgG has an amino acid sequence of sequence number: 38 (i.e., an amino acid sequence having 100% sequence similarity to sequence number: 38).

[0058] According to certain embodiments, the CDR-L1, CDR-L2 and CDR-L3 of p30-077 scFv or p30-077 IgG have the amino acid sequences of sequence numbers 7-9, respectively, and the CDR-H1, CDR-H2 and CDR-H3 of p30-077 scFv or p30-077 IgG have the amino acid sequences of sequence numbers 10-12, respectively. According to certain embodiments, the VL domain of p30-077 scFv or p30-077 IgG comprises an amino acid sequence having at least 85% sequence similarity to sequence number 39; and the VH domain of p30-077 scFv or p30-077 IgG comprises an amino acid sequence having at least 85% sequence similarity to sequence number 40. According to a preferred embodiment, the VL domain and VH domain of p30-077 scFv or p30-077 IgG respectively comprise an amino acid sequence having at least 90% sequence similarity to sequence numbers: 39 and 40. More preferably, the VL domain and VH domain of p30-077 scFv or p30-077 IgG respectively comprise an amino acid sequence having at least 95% sequence similarity to sequence numbers: 39 and 40. In an operational embodiment of the present disclosure, the VL domain of p30-077 scFv or p30-077 IgG has the amino acid sequence of sequence number: 39, and the VH domain of p30-077 scFv or p30-077 IgG has the amino acid sequence of sequence number: 40.

[0059] According to certain embodiments, the CDR-L1, CDR-L2 and CDR-L3 of p30-090 scFv or p30-090 IgG have the amino acid sequences of sequence numbers 13-15, respectively, and the CDR-H1, CDR-H2 and CDR-H3 of p30-090 scFv or p30-090 IgG have the amino acid sequences of sequence numbers 16-18, respectively. According to certain embodiments, the VL domain of p30-090 scFv or p30-090 IgG comprises an amino acid sequence having at least 85% sequence similarity to sequence number 41; and the VH domain of p30-090 scFv or p30-090 IgG comprises an amino acid sequence having at least 85% sequence similarity to sequence number 42. According to a preferred embodiment, the VL domain and VH domain of p30-090 scFv or p30-090 IgG respectively comprise an amino acid sequence having at least 90% sequence similarity to sequence numbers: 41 and 42. More preferably, the VL domain and VH domain of p30-090scFv or p30-090IgG respectively comprise an amino acid sequence having at least 95% sequence similarity to sequence numbers: 41 and 42. In an operational embodiment of the present disclosure, the VL domain of p30-090 scFv or p30-090 IgG has an amino acid sequence of sequence number: 41, and the VH domain of p30-090 scFv or p30-090 IgG has an amino acid sequence of sequence number: 42.

[0060] According to certain embodiments, the CDR-L1, CDR-L2 and CDR-L3 of p30-091 scFv or p30-091 IgG have the amino acid sequences of sequence numbers 19-21, respectively, and the CDR-H1, CDR-H2 and CDR-H3 of p30-091 scFv or p30-091 IgG have the amino acid sequences of sequence numbers 22-24, respectively. According to certain embodiments, the VL domain of p30-091 scFv or p30-091 IgG comprises an amino acid sequence having at least 85% sequence similarity to sequence number 43; and the VH domain of p30-091 scFv or p30-091 IgG comprises an amino acid sequence having at least 85% sequence similarity to sequence number 44. According to a preferred embodiment, the VL domain and VH domain of p30-091 scFv or p30-091 IgG respectively comprise an amino acid sequence having at least 90% sequence similarity to sequence numbers: 43 and 44. More preferably, the VL domain and VH domain of p30-091 scFv or p30-091 IgG respectively comprise an amino acid sequence having at least 95% sequence similarity to sequence numbers: 43 and 44. In an operational embodiment of the present disclosure, the VL domain of p30-091 scFv or p30-091 IgG has an amino acid sequence of sequence number: 43, and the VH domain of p30-091 scFv or p30-091 IgG has an amino acid sequence of sequence number: 44.

[0061] According to certain embodiments, the CDR-L1, CDR-L2 and CDR-L3 of p30-096 scFv or p30-096 IgG have the amino acid sequences of sequence numbers 25-27, respectively, and the CDR-H1, CDR-H2 and CDR-H3 of p30-096 scFv or p30-096 IgG have the amino acid sequences of sequence numbers 28-30, respectively. According to certain embodiments, the VL domain of p30-096 scFv or p30-096 IgG comprises an amino acid sequence having at least 85% sequence similarity to sequence number 45; and the VH domain of p30-096 scFv or p30-096 IgG comprises an amino acid sequence having at least 85% sequence similarity to sequence number 46. According to a preferred embodiment, the VL domain and VH domain of p30-096 scFv or p30-096 IgG respectively comprise an amino acid sequence having at least 90% sequence similarity to sequence numbers: 45 and 46. More preferably, the VL domain and VH domain of p30-096 scFv or p30-096 IgG respectively comprise an amino acid sequence having at least 95% sequence similarity to sequence numbers: 45 and 46. In an operational embodiment of the present disclosure, the VL domain of p30-096 scFv or p30-096 IgG has an amino acid sequence of sequence number: 45, and the VH domain of p30-096 scFv or p30-096 IgG has an amino acid sequence of sequence number: 46.

[0062] According to alternative embodiments, the CDR-L1, CDR-L2 and CDR-L3 of p30-097 scFv or p30-097 IgG have amino acid sequences of sequence numbers: 31-33, respectively, and the CDR-H1, CDR-H2 and CDR-H3 of p30-097 scFv or p30-097 IgG have amino acid sequences of sequence numbers: 34-36, respectively. According to certain embodiments, the VL domain of p30-097 scFv or p30-097 IgG comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 47; and the VH domain of p30-097 scFv or p30-097 IgG comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 48. According to a preferred embodiment, the VL domain and VH domain of p30-097 scFv or p30-097 IgG respectively comprise an amino acid sequence having at least 90% sequence similarity to sequence numbers: 47 and 48. More preferably, the VL domain and VH domain of p30-097scFv or p30-097IgG respectively comprise an amino acid sequence having at least 95% sequence similarity to sequence numbers: 47 and 48. In an operational embodiment of the present disclosure, the VL domain of p30-097scFv or p30-097 IgG has an amino acid sequence of sequence number: 47, and the VH domain of p30-097 scFv or p30-097 IgG has an amino acid sequence of sequence number: 48.

[0063] (II-4) Use of antibody fragments and recombinant antibodies in the diagnosis of ASFV infection

[0064] According to certain embodiments of the present disclosure, each antibody fragment and recombinant antibody can be used to detect ASFV; accordingly, the antibody fragment or recombinant antibody of the present invention can be used as a detection reagent for detecting ASFV infection.

[0065] Therefore, another aspect of the present disclosure provides a kit for detecting ASFV infection in an individual (i.e., a pig). The kit comprises at least a first antibody, a second antibody, and a container containing the first and second antibodies. Depending on the desired purpose, the first and second antibodies are individually selected from antibody fragments and recombinant antibodies as described in sections (II-1) to (II-3) of the present disclosure. The kit of the present disclosure can be used in any detection technique known to those skilled in the art (e.g., ELISA, LFIA, SPR, Western blot, and flow cytometer) to detect ASFV infection in a biological sample. According to certain operational embodiments, the first and second antibody fragments / recombinant antibodies of the kit of the present disclosure are used as capture antibodies and detection antibodies, respectively, in ELISA, Western blot, or LFIA.

[0066] In one embodiment, the kit is named "D96C77", which contains p30-077 IgG as the first antibody and p30-096 IgG as the second antibody. In another embodiment, the kit is named "D91C90", which contains p30-090 IgG as the first antibody and p30-091 IgG as the second antibody. In another embodiment, the kit is named "D97C52", which contains p30-052 IgG as the first antibody and p30-097 IgG as the second antibody.

[0067] Optionally, the kit may further comprise instructions for use, instructing the user on how to use the antibody fragment or recombinant antibody to detect ASFV infection.

[0068] In addition, the present disclosure also encompasses a method for diagnosing whether an individual is infected with ASFV by using a biological sample isolated from the individual. The method comprises using the antibody fragment, recombinant antibody or kit of the present disclosure to detect whether there is a viral protein of ASFV in the biological sample, wherein when the viral protein (e.g., p30 protein) is present in the biological sample, the individual is diagnosed as being infected with ASFV.

[0069] Non-limiting examples of biological samples suitable for the methods of the present invention include whole blood samples, serum, plasma, kidney, liver, lung, spleen, lymph nodes, spleen, tonsils and small intestine of an individual. Alternatively, the biological sample can be a swab sample from the individual's oral cavity, nasal cavity or anus.

[0070] Based on the above diagnosis results, a person skilled in the art or a clinical practitioner can administer an effective amount of anti-viral therapy to an individual in need (i.e., an ASFV-infected pig) to reduce and / or alleviate the symptoms associated with ASFV infection. Examples of anti-viral therapies suitable for the methods of the present disclosure include, but are not limited to, cilantro, enflurane carboxylic acid, grifloxacin, balofloxacin, tofloxacin, gatifloxacin, gareoxacin, or a combination thereof. Alternatively, an individual diagnosed as being infected with ASFV (i.e., an ASFV-infected pig) is promptly separated from other livestock on the farm to prevent and control the spread of ASFV.

[0071] A number of embodiments are presented below to illustrate certain aspects of the present disclosure, so that those with ordinary knowledge in the art to which the present invention belongs can practice the present invention. These embodiments should not be considered as limiting the scope of the present invention. It is believed that those with ordinary knowledge in the art to which the present invention belongs can fully utilize and practice the present invention without excessive interpretation after reading the description presented herein. All public documents cited herein are hereby incorporated by reference in their entirety. Example

[0072] Materials and Methods

[0073] Cell lines

[0074] Suspension Expi293FTM cells were cultured in Expi293TM expression medium, shaken at 110 rpm, and placed in an incubator at 37°C with 8% CO2.

[0075] Confirmation of IgG1 selected from phage-expressed synthetic scFv antibody library

[0076] The synthetic scFv antibody library expressed by phage was constructed and confirmed according to the same process described in PCT applications PCT / US2016 / 19128 and PCT / US2018 / 56627. The relevant experimental steps for panning the phage-expressed antibody library, screening the phage-expressed scFv binders, confirming the scFv binding to the cognate antigen and protein A / L by ELISA, reformatting the scFv to IgG1, and expressing and purifying IgG are all disclosed in the contents of PCT applications PCT / US2016 / 19128 and PCT / US2018 / 56627.

[0077] Detection of p30 protein by immunoblotting

[0078] 250 ng of recombinant p30 protein was separated by 12% dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to polyvinylidene difluoride (PVDF) membrane and detected with specific IgG antibody (1 μg / mL). Specific bands were detected by antibody conjugated with horseradish peroxidase (HRP) (1 mg / mL, 1:2,500 dilution), and the signals were displayed with chemiluminescent substrate and chemiluminescent imaging system.

[0079] EC 50 Detecting Antibody-Antigen Interactions

[0080] EC of IgG was determined by the titer of IgG antibody against immobilized recombinant p30 protein in ELISA 50Specifically, p30 protein (0.5 μg per well) was diluted with PBS buffer (pH 7.4) and coated on a 96-well immunoculture plate, followed by blocking with 5% skim milk in PBST for 1 hour. At the same time, the antibody was serially diluted 2-fold with PBST containing 5% skim milk (stock concentration: 0.5 μg / ml) to produce 11 different concentrations of antibodies. After the blocking step, 100 μl of the diluted antibody sample was added to each well and shaken for 1 hour. Each well was washed three times with 300 μl of PBST, and then 100 μl of HRP-conjugated goat anti-human IgG antibody (diluted 1:5000 in PBST containing 5% skim milk) was added for one hour. Each well was washed three times with 300 μl of PBST buffer and then twice with 300 μl of PBS. The wells were then developed with 3,3'5,5'-tetramethylbenzidine (TMB) for 5 minutes and the reaction was terminated with 1.0 M HCl. The absorbance at a wavelength of 450 nm was measured.

[0081] Detection of p30 protein by sandwich ELISA

[0082] The capture antibody (1 μg per well) diluted in PBS buffer (pH 7.4) was coated on a 96-well immunoculture plate and reacted overnight. 5% skim milk dissolved in PBST was added to the culture plate for blocking for 1 hour. Then, p30 protein (stock concentration: 1 μg / ml) diluted 2 times was added to the culture plate (100 μl per well) and reacted at room temperature for 1 hour. Each well was washed three times with 300 μl of PBST, and then 100 μl of detection antibody diluted in PBST containing 5% skim milk (dilution ratio of 1:2000) was added and reacted at room temperature for 1 hour. After washing each well three times with 300 μl of PBST buffer, it was washed twice with 300 μl of PBS, and then the color was developed with TMB substrate for 5 minutes, and the reaction was terminated by 1.0 M HCl, and finally the absorbance at a wavelength of 450 nm was measured.

[0083] Preparation of AL2C and IgG bonded to colloidal gold

[0084] Mix 50 μl of 0.2M K2CO3 (pH 11.5) and 10 ml of colloidal gold solution (pH 5-6) to adjust the pH (final pH 8), then add 500 g of IgG or 167 g of AL2C to the colloidal gold solution and react at room temperature for 40 minutes. Add 1 ml of blocking buffer (10% bovine serum albumin (BSA) dissolved in 20 mM sodium borate, pH 9.3) and react at room temperature for 15 minutes to stop the reaction, then centrifuge (15,000 x g, 30 minutes, 4°C) the reaction. Pour off the supernatant, and resuspend the precipitate in 10 ml of washing buffer (1% BSA dissolved in 20 mM sodium borate, pH 9.3), then centrifuge (15,000 x g, 30 minutes, 4°C) the suspension. This washing step was repeated twice, and the pellet was resuspended in 1 ml of 1% BSA (pH 9.3) dissolved in 20 mM sodium borate to prepare a conjugate pad.

[0085] Assembling LFIA strips

[0086] Using a lateral flow dispenser driven by a syringe infusion pump, 1 μg of capture antibody, antigen or AL2C in PBS buffer solution was applied to form strips on a nitrocellulose membrane at intervals of 1 cm. Other nitrocellulose membranes with immobilized antigen or capture antibody, conjugate pads and sample pads were prepared according to the above method, and LFIA strips were assembled.

[0087] Example 1 Confirmation of recombinant antibodies

[0088] The p30 protein of ASFV was synthesized, and anti-p30 antibodies were screened according to the methods described in PCT applications PCT / US2016 / 19128 and PCT / US2018 / 56627.

[0089] Six anti-p30 scFvs screened from the scFv antibody library expressed by phages were reconstituted into IgG1, and these IgG1s were named "p30-052 IgG", "p30-077 IgG", "p30-090IgG", "p30-091 IgG", "p30-096IgG" and "p30-097 IgG", respectively. The VL domain and VH domain sequences of these antibodies are summarized in Table 1. According to the analysis results of ELISA, the binding affinity (EC) of p30-077, p30-096, p30-090, p30-091, p30-052 and p30-097 IgG to the p30 protein 50 ) were 2.6, 2.5, 0.9, 2.6, 2 and 1.2 ng / mL respectively.

[0090] Table 1 VL domain and VH domain sequences of specific antibodies *CDR sequences are indicated in bold, including the three CDRs in the VL domain (i.e., CDR-L1, CDR-L2, and CDR-L3 from N-terminus to C-terminus), and the three CDRs in the VH domain (i.e., CDR-H1, CDR-H2, and CDR-H3 from N-terminus to C-terminus).

[0091] The binding affinity of p30-077, p30-096, p30-090, p30-091, p30-052 and p30-097 IgG to the p30 protein of ASFV was further tested by sandwich ELISA. As shown in the data summarized in Table 2, the antibodies of the present invention can be used as detection antibodies and capture antibodies for detecting ASFV, wherein the EC of antibody pair D96C77 to ASFV is 50 The EC value of antibody D91C90 against ASFV was 16.52 ng / mL. 50 The EC value of antibody D97C52 against ASFV is 8.91 ng / mL. 50 It is 8.43 ng / mL.

[0092] Table 2 Sandwich ELISA EC of specific antibodies against p30 protein of ASFV 50

[0093] In addition to being used in ELISA, the antibodies of the present disclosure can also be used as detection antibodies for ASFV in Western blot. According to the experimental results, p30-007, p30-011, p30-014, p30-015, p30-019 and p30-020 IgG can specifically recognize p30 protein, but not bind to p54 protein (another structural protein of ASFV) (data not shown).

[0094] Example 2 Construction of LFIA device for detecting ASFV

[0095] To construct the LFIA device, six anti-p30 antibodies were conjugated to colloidal gold according to the procedures described in "Materials and Methods" of the present disclosure. The analysis results indicated that the antibodies of the present disclosure could efficiently conjugate to colloidal gold (data not shown).

[0096] In this example, three LFIA device prototypes were established, wherein the LFIA device prototype D96C77 used p30-077 IgG as a capture antibody and p30-096 IgG as a detection antibody bound to colloidal gold; the LFIA device prototype D91C90 used p30-090 IgG as a capture antibody and p30-091 IgG as a detection antibody bound to colloidal gold; and the LFIA device prototype D97C52 used p30-052 IgG as a capture antibody and p30-097 IgG as a detection antibody bound to colloidal gold. The detection limit of each LFIA device prototype was determined using two samples containing the p30 protein of ASFV. The first sample contained a sample of Expi293 TM cells-expressed p30 protein, and a second sample contained p30 protein expressed by E. coli. Figures 1A to 1C The data in panel (A) show that each antibody has a significant effect on D96C77 ( Figure 1A ), antibody to D91C90( Figure 1B ) and antibodies against D97C52( Figure 1C ) can be used with Expi293 TM The detection limit of cell-expressed p30 protein binding was between 1.0-7.8 ng / assay. Figures 1A to 1C The data in panel (B) further confirmed that each antibody pair interacted with Expi293 TM The p30 protein expressed by cells has binding specificity.

[0097] in accordance with Figures 2A to 2C The data in panel (A) show that each antibody has a significant effect on D96C77 ( Figure 2A ), antibody to D91C90( Figure 2B ) and antibodies against D97C52( Figure 2C ) were able to bind to E. coli-expressed p30 protein with a detection limit ranging from 1.0 to 3.9 ng / assay. Figures 2A to 2C The data in panel (B) also demonstrate that each antibody has binding affinity to E. coli-expressed p30 protein.

[0098] In summary, the antibodies screened from the GH synthetic antibody library were confirmed to have high binding affinity and specificity to the p30 protein of ASFV. The antibodies (including 6 anti-p30 IgGs) derived from the GH antibody library of the present disclosure can be used to detect the corresponding viral proteins in sandwich ELISA and LFIA without further affinity maturation, and the detection limit is 1 ng / test. Accordingly, the antibodies of the present disclosure can be used to detect ASFV, thereby preventing and controlling the spread of the infectious disease.

[0099] It should be understood that the description of the aforementioned embodiments is given by way of example only, and various modifications may be made by those with ordinary knowledge in the technical field to which the present invention belongs. The above description, embodiments and experimental results provide a complete description of the structure and use of exemplary embodiments of the present invention. Although specific embodiments of the present invention are disclosed in the above embodiments, they are not intended to limit the present invention. Those with ordinary knowledge in the technical field to which the present invention belongs may make various changes and modifications to the same without departing from the principles and spirit of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of the accompanying patent application. Sequence Listing <110> Zhou Meiyin <120> Recombinant antibody, kit containing the recombinant antibody and use thereof in diagnosing African swine fever virus <130> P4157-PCT <160> 53 <170> BiSSAP 1.3 <210> 1 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-052_CDR-L1 <400> 1 Arg Ala Ser Gln Asp Val Asp Asn Asn Val Ala 1 5 10 <210> 2 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-052_CDR-L2 <400> 2 Ser Ser Pro Gly Phe Leu Tyr Ser 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-052_CDR-L3 <400> 3 Gln Gln Tyr Phe Asn Phe Pro Ile Thr 1 5 <210> 4 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-052_CDR-H1 <400> 4 Ala Ala Ser Gly Phe Thr Ile Asp Ser Gly Phe Ile His 1 5 10 <210> 5 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-052_CDR-H2 <400> 5 Gly Ile Gly Pro Phe Trp Gly Phe Thr Phe 1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic __p30-052_CDR-H3 <400> 6 Ala Arg Tyr Gly Ser Ser Ser Tyr Gly Phe Asp Tyr 1 5 10 <210> 7 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-077_CDR-L1 <400> 7 Arg Ala Ser Gln Asp Val Asn Asn Asn Val Ala 1 5 10 <210> 8 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-077_CDR-L2 <400> 8 Ser Ser Pro Thr Tyr Leu Tyr Ser 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-077_CDR-L3 <400> 9 Gln Gln Trp Tyr Asp Trp Pro Ile Thr 1 5 <210> 10 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-077_CDR-H1 <400> 10 Ala Ala Ser Gly Phe Thr Ile Gly Asp Trp Gly Ile His 1 5 10 <210> 11 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-077_CDR-H2 <400> 11 Gly Ile Trp Pro Phe Gly Gly Phe Thr Phe 1 5 10 <210> 12 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-077_CDR-H3 <400> 12 Ala Arg Gly Ser Phe Tyr Trp Asp Tyr 1 5 <210> 13 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-090_CDR-L1 <400> 13 Arg Ala Ser Gln Asp Val Asn Asn Asp Val Ala 1 5 10 <210> 14 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-090_CDR-L2 <400> 14 Phe Tyr Pro Gly Gly Leu Tyr Ser 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-090_CDR-L3 <400> 15 Gln Gln Ser Trp Asn Tyr Pro Leu Thr 1 5 <210> 16 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-090_CDR-H1 <400> 16 Ala Ala Ser Gly Phe Thr Ile Asp Gly Gly Gly Ile His 1 5 10 <210> 17 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-090_CDR-H2 <400> 17 Gly Ile Trp Pro Ser Trp Gly Tyr Thr Tyr 1 5 10 <210> 18 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-090_CDR-H3 <400> 18 Ala Arg Gly Phe Tyr Trp Tyr Asp Tyr 1 5 <210> 19 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-091_CDR-L1 <400> 19 Lys Ser Asn Gln Asn Leu Leu Asp Ser Gly Asp Gln Gly Thr Tyr Leu 1 5 10 15 Asn <210> 20 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-091_CDR-L2 <400> 20 Ser Asp Val Ser Asn Leu Tyr Ser 1 5 <210> twenty one <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-091_CDR-L3 <400> twenty one Tyr Gln Ala Thr Ser Phe Pro Phe Thr 1 5 <210> twenty two <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-091_CDR-H1 <400> twenty two Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr Trp Ile His 1 5 10 <210> twenty three <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-091_CDR-H2 <400> twenty three Tyr Ile Asn Pro His Tyr Gly Leu Thr Asp 1 5 10 <210> twenty four <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-091_CDR-H3 <400> twenty four Ala Arg Gly Trp Leu Leu Asp Tyr 1 5 <210> 25 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-096_CDR-L1 <400> 25 Ser Gly Ser Ser Ser Asn Ile Gly Asp Asn Asp Val Tyr Trp Tyr 1 5 10 15 <210> 26 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-096_CDR-L2 <400> 26 Tyr Gly Thr Thr Ser Leu Tyr Ser 1 5 <210> 27 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-096_CDR-L3 <400> 27 Ala Ala Gly Gly Trp Gly His Asn Gly Val 1 5 10 <210> 28 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-096_CDR-H1 <400> 28 Ala Ala Ser Gly Phe Thr Ile Ser Asp Trp Ser Ile His Trp Val 1 5 10 15 <210> 29 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-096_CDR-H2 <400> 29 Gly Ile Trp Pro Tyr Trp Gly Phe Thr Ser 1 5 10 <210> 30 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-096_CDR-H3 <400> 30 Ala Arg Phe Val Ser Tyr Tyr Gly Tyr Ser Leu Met Asp Tyr 1 5 10 <210> 31 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-097_CDR-L1 <400> 31 Lys Ser Asn Gln Asn Leu Leu His Ser Asp Gly Gly Thr Ser Leu His 1 5 10 15 TrpTyr <210> 32 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-097_CDR-L2 <400> 32 Phe Asp Thr Ser Asn Leu Tyr Ser 1 5 <210> 33 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-097_CDR-L3 <400> 33 Tyr Gln Thr Ser Arg Leu Pro Gly 1 5 <210> 34 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-097_CDR-H1 <400> 34 Lys Ala Ser Gly Tyr Thr Phe Thr Thr Phe Trp Met Tyr Trp Val 1 5 10 15 <210> 35 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-097_CDR-H2 <400> 35 Gly Ile Asp Pro His His Gly Leu Thr Tyr 1 5 10 <210> 36 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-097_CDR-H3 <400> 36 Ala Arg Gly Asp Trp Asp Gly Leu Met Asp Tyr 1 5 10 <210> 37 <211> 110 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-052_VL <400> 37 Met Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asp 20 25 30 Asn Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Ser Ser Pro Gly Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Asn Phe 85 90 95 Pro Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 <210> 38 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-052_VH <400> 38 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asp Ser Gly 20 25 30 Phe Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Gly Pro Phe Trp Gly Phe Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Gly Ser Ser Ser Tyr Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 115 120 <210> 39 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Synthetic_p30-077_VL <400> 39 Met Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn 20 25 30 Asn Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Ser Ser Pro Thr Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Tyr Asp Trp 85 90 95 Pro Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 <210> 40 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic_p30-077_VH <400> 40 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Gly Asp Trp 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Trp Pro Phe Gly Gly Phe Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Phe Tyr Trp Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val See See Ala See Ala Ala Ala 115 120 <210> 41 <211> 110 <212> PRT <213> artificial sequence <220> <223> Synthesis_p30-090_VL <400> 41 Met Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn 20 25 30 Asn Asp Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Phe Tyr Pro Gly Gly Leu Tyr Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Trp Asn Tyr 85 90 95 Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 <210> 42 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic_p30-090_VH <400> 42 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Asp Gly Gly 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Trp Pro Ser Trp Gly Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Phe Tyr Trp Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val See See Ala See Ala Ala Ala 115 120 <210> 43 <211> 116 <212> PRT <213> artificial sequence <220> <223> Synthesis_p30-091_VL <400> 43 Met Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ser Asn Gln Asn Leu Leu 20 25 30 Asp Ser Gly Asp Gln Gly Thr Tyr Leu Asn Trp Tyr Gln Gln Lys Pro 35 40 45 Gly Lys Ala Pro Lys Leu Leu Ile Ser Asp Val Ser Asn Leu Tyr Ser 50 55 60 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70 75 80 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 85 90 95 Tyr Gln Ala Thr Ser Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val 100 105 110 Glu Ile Lys Arg 115 <210> 44 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic_p30-091_VH <400> 44 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Asn Pro His Tyr Gly Leu Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Leu Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Ala Ala Ala 115 120 <210> 45 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-096_VL <400> 45 Met Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Ser Gly Ser Ser Ser Asn Ile 20 25 30 Gly Asp Asn Asp Val Tyr Trp Tyr Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Gly Thr Thr Ser Leu Tyr Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Ala 85 90 95 Gly Gly Trp Gly His Asn Gly Val Phe Gly Gln Gly Thr Lys Val Glu 100 105 110 Ile Lys Arg 115 <210> 46 <211> 128 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-096_VH <400> 46 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Trp 20 25 30 Ser Ile His Trp Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Gly Ile Trp Pro Tyr Trp Gly Phe Thr Ser Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Phe Val Ser Tyr Tyr Gly Tyr Ser Leu Met Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 115 120 125 <210> 47 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-097_VL <400> 47 Met Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ser Asn Gln Asn Leu Leu 20 25 30 His Ser Asp Gly Gly Thr Ser Leu His Trp Tyr Trp Tyr Gln Gln Lys 35 40 45 Pro Gly Lys Ala Pro Lys Leu Leu Ile Phe Asp Thr Ser Asn Leu Tyr 50 55 60 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 65 70 75 80 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 85 90 95 Cys Tyr Gln Thr Ser Arg Leu Pro Gly Phe Gly Gln Gly Thr Lys Val 100 105 110 Glu Ile Lys Arg 115 <210> 48 <211> 125 <212> PRT <213> Artificial sequence <220> <223> Synthetic_p30-097_VH <400> 48 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Phe 20 25 30 Trp Met Tyr Trp Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Gly Ile Asp Pro His His Gly Leu Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Gly Asp Trp Asp Gly Leu Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Ala Ala Ala 115 120 125 <210> 49 <211> 181 <212> PRT <213> Artificial Sequence <220> <223> Synthetic_p30 Protein <400> 49 Met Lys Met Glu Val Ile Phe Lys Thr Asp Leu Arg Ser Ser Ser Gln 1 5 10 15 Val Val Phe His Ala Gly Ser Leu Tyr Asn Trp Phe Ser Val Glu Ile 20 25 30 Ile Asn Ser Gly Arg Ile Val Thr Thr Ala Ile Lys Thr Leu Leu Ser 35 40 45 Thr Val Lys Tyr Asp Ile Val Lys Ser Ala Arg Ile Tyr Ala Gly Gln 50 55 60 Gly Tyr Thr Glu His Gln Ala Gln Glu Glu Trp Asn Met Ile Leu His 65 70 75 80 Val Leu Phe Glu Glu Glu Thr Glu Ser Ser Ala Ser Ser Glu Asn Ile 85 90 95 His Glu Lys Asn Asp Asn Glu Thr Asn Glu Cys Thr Ser Ser Phe Glu 100 105 110 Thr Leu Phe Glu Gln Glu Pro Ser Ser Glu Val Pro Lys Asp Ser Lys 115 120 125 Leu Tyr Met Leu Ala Gln Lys Thr Val Gln His Ile Glu Gln Tyr Gly 130 135 140 Lys Ala Pro Asp Phe Asn Lys Val Ile Arg Ala His Asn Phe Ile Gln 145 150 155 160 Thr Ile Tyr Gly Thr Pro Leu Lys Glu Glu Glu Lys Glu Val Val Arg 165 170 175 Leu Met Val Ile Lys 180 <210> 50 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Synthetic _F primer <400> 50 cgtgtcgcat ctgaagtgca gctggtggaa tcggga 36 <210> 51 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic _R primer <400> 51 gaccgatggg cccttggtgc tagccgagct cacggtaaca agggtgcc 48 <210> 52 <211> 59 <212> DNA <213> Artificial sequence <220> <223> Synthetic _F primer <400> 52 caggtgcacg atgtgatggt accgatattc aaatgaccca gagcccgagc agcctgagc 59 <210> 53 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Synthetic _R primer <400> 53 tgcagccacc gtacgtttga tttccacctt ggtgcc 36

Claims

1. A recombinant antibody or a fragment thereof, comprising a variable light chain (VL) domain and a variable heavy chain (VH) domain, wherein The VL domain comprises the amino acid sequence of sequence numbering: 1-3, and the VH domain comprises the amino acid sequence of sequence numbering: 4-6; The VL domain comprises the amino acid sequence of sequence numbers: 7-9, and the VH domain comprises the amino acid sequence of sequence numbers: 10-12; The VL domain comprises the amino acid sequence of sequence numbers: 13-15, and the VH domain comprises the amino acid sequence of sequence numbers: 16-18; The VL domain comprises the amino acid sequence of sequence numbers: 19-21, and the VH domain comprises the amino acid sequence of sequence numbers: 22-24; The VL domain comprises the amino acid sequence of sequence numbering: 25-27, and the VH domain comprises the amino acid sequence of sequence numbering: 28-30; or The VL domain comprises the amino acid sequence of sequence numbers: 31-33, and the VH domain comprises the amino acid sequence of sequence numbers: 34-36.

2. The recombinant antibody according to claim 1, wherein The VL domain comprises an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 37, and the VH domain comprises an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 38; The VL domain comprises an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 39, and the VH domain comprises an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 40; The VL domain comprises an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 41, and the VH domain comprises an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 42; The VL domain comprises an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 43, and the VH domain comprises an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 44; The VL domain comprises an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 45, and the VH domain comprises an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 46; or The VL domain comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 47, and the VH domain comprises an amino acid sequence having at least 85% sequence similarity to sequence number:

48.

3. The recombinant antibody according to claim 2, wherein The VL domain comprises an amino acid sequence having 100% sequence similarity to SEQ ID NO: 37, and the VH domain comprises an amino acid sequence having 100% sequence similarity to SEQ ID NO: 38; The VL domain comprises an amino acid sequence having 100% sequence similarity to SEQ ID NO: 39, and the VH domain comprises an amino acid sequence having 100% sequence similarity to SEQ ID NO: 40; The VL domain comprises an amino acid sequence having 100% sequence similarity to SEQ ID NO: 41, and the VH domain comprises an amino acid sequence having 100% sequence similarity to SEQ ID NO: 42; The VL domain comprises an amino acid sequence having 100% sequence similarity to SEQ ID NO: 43, and the VH domain comprises an amino acid sequence having 100% sequence similarity to SEQ ID NO: 44; The VL domain comprises an amino acid sequence having 100% sequence similarity to SEQ ID NO: 45, and the VH domain comprises an amino acid sequence having 100% sequence similarity to SEQ ID NO: 46; or The VL domain comprises an amino acid sequence having 100% sequence similarity with sequence number: 47, and the VH domain comprises an amino acid sequence having 100% sequence similarity with sequence number:

48.

4. A kit for detecting African swine fever virus (ASFV), comprising a first recombinant antibody and a second recombinant antibody, each of which is selected from the recombinant antibody of claim 1; and a container, comprising the first recombinant antibody and the second recombinant antibody.

5. The kit of claim 4, wherein The VL domain of the first recombinant antibody comprises the amino acid sequence of sequence numbers: 1-3, and the VH domain of the first recombinant antibody comprises the amino acid sequence of sequence numbers: 4-6; and The VL domain of the second recombinant antibody comprises the amino acid sequence of sequence numbers: 31-33, and the VH domain of the second recombinant antibody comprises the amino acid sequence of sequence numbers: 34-36.

6. The kit of claim 5, wherein The VL domain of the first recombinant antibody comprises an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 37, and the VH domain of the first recombinant antibody comprises an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 38; and The VL domain of the second recombinant antibody comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 47, and the VH domain of the second recombinant antibody comprises an amino acid sequence having at least 85% sequence similarity to sequence number:

48.

7. The kit of claim 6, wherein The VL domain of the first recombinant antibody comprises an amino acid sequence having 100% sequence similarity to SEQ ID NO: 37, and the VH domain of the first recombinant antibody comprises an amino acid sequence having 100% sequence similarity to SEQ ID NO: 38; and The VL domain of the second recombinant antibody comprises an amino acid sequence having 100% sequence similarity to sequence number: 47, and the VH domain of the first recombinant antibody comprises an amino acid sequence having 100% sequence similarity to sequence number:

48.

8. The kit of claim 4, wherein The VL domain of the first recombinant antibody comprises the amino acid sequence of sequence numbers: 7-9, and the VH domain of the first recombinant antibody comprises the amino acid sequence of sequence numbers: 10-12; and The VL domain of the second recombinant antibody comprises the amino acid sequence of sequence numbers: 25-27, and the VH domain of the second recombinant antibody comprises the amino acid sequence of sequence numbers: 28-30.

9. The kit of claim 8, wherein The VL domain of the first recombinant antibody comprises an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 39, and the VH domain of the first recombinant antibody comprises an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 40; and The VL domain of the second recombinant antibody comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 45, and the VH domain of the second recombinant antibody comprises an amino acid sequence having at least 85% sequence similarity to sequence number:

46.

10. The kit of claim 9, wherein The VL domain of the first recombinant antibody comprises an amino acid sequence having 100% sequence similarity to SEQ ID NO: 39, and the VH domain of the first recombinant antibody comprises an amino acid sequence having 100% sequence similarity to SEQ ID NO: 40; and The VL domain of the second recombinant antibody comprises an amino acid sequence having 100% sequence similarity to sequence number: 45, and the VH domain of the first recombinant antibody comprises an amino acid sequence having 100% sequence similarity to sequence number:

46.

11. The kit of claim 4, wherein The VL domain of the first recombinant antibody comprises the amino acid sequence of sequence numbers: 13-15, and the VH domain of the first recombinant antibody comprises the amino acid sequence of sequence numbers: 16-18; and The VL domain of the second recombinant antibody comprises the amino acid sequence of sequence numbers: 19-21, and the VH domain of the second recombinant antibody comprises the amino acid sequence of sequence numbers: 22-24.

12. The kit of claim 11, wherein The VL domain of the first recombinant antibody comprises an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 41, and the VH domain of the first recombinant antibody comprises an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 42; and The VL domain of the second recombinant antibody comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 43, and the VH domain of the second recombinant antibody comprises an amino acid sequence having at least 85% sequence similarity to sequence number:

44.

13. The kit of claim 12, wherein The VL domain of the first recombinant antibody comprises an amino acid sequence having 100% sequence similarity to SEQ ID NO: 41, and the VH domain of the first recombinant antibody comprises an amino acid sequence having 100% sequence similarity to SEQ ID NO: 42; and The VL domain of the second recombinant antibody comprises an amino acid sequence having 100% sequence similarity to sequence number: 43, and the VH domain of the first recombinant antibody comprises an amino acid sequence having 100% sequence similarity to sequence number:

44.

14. A method for confirming whether an individual is infected with ASFV by using a biological sample isolated from the individual, comprising using the recombinant antibody according to claim 1 or the kit according to claim 4 to detect whether the viral protein of the ASFV is present in the biological sample, wherein the presence of the viral protein indicates that the individual is infected with ASFV.

15. The method of claim 14, wherein the viral protein is a 30-kDa phosphoprotein.

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