AU2020103187A4

AU2020103187A4 - A Monoclonal Antibody Capable Of Competing With Positive Serum For Binding To African Swine Fever Virus B646L Antigen And The Application Thereof

Info

Publication number: AU2020103187A4
Application number: AU2020103187A
Authority: AU
Inventors: Chunyan FENG; Xiangmei LIN; Xiaofei Liu; Songyin QIU; Caixia Wang; Qin Wang; Shaoqiang WU
Original assignee: Chinese Academy of Inspection and Quarantine CAIQ
Current assignee: Chinese Academy of Inspection and Quarantine CAIQ
Priority date: 2020-09-09
Filing date: 2020-11-02
Publication date: 2021-01-14
Anticipated expiration: 2028-11-02
Also published as: CN112111005A; CN112111005B

Abstract

The invention discloses an antigen binding protein, an antibody or an active fragment capable of specifically and competitively binding an African swine fever virus B646L antigen with positive serum. The African swine fever virus competitive ELISA detection kit comprising the antigen binding protein, the antibody or the active fragment, and its preparation method and application was also included. The African swine fever virus competitive ELISA detection kit in this invention has high sensitivity and good specificity, and can be used for detecting specific antibodies of African swine fever virus. The competitive ELISA detection kit can specifically compete with antibodies for binding to B646L antigen in serum, and has the advantages of rapidness, convenience, sensitivity, specificity and the like in the clinical detection of African swine fever antibodies.

Description

A monoclonal antibody capable of competing with positive serum

for binding to African swine fever virus B646L antigen and the

application thereof

Technical Field The invention belongs to the biological field, and particularly involves a monoclonal antibody which can compete with positive serum for binding to African swine fever virus B646L antigen and its application in detecting the African swine fever virus antibody in a competitive ELISA detection method.

Background Art .0 African swine fever (ASF) was an acute and highly lethal infectious disease in pigs caused by African swine fever virus (ASFV). ASF was discovered in Kenya in 1921, and has been popular in sub-Saharan African countries since then. It has caused widespread epidemic in some countries in Western Europe and Latin America, and resulted in serious social and economic losses. Since it .5 was first reported in Shenyang in August 2018, it has spread rapidly to Shandong, Henan, Jiangsu, Hebei, Anhui, Beijing and other provinces. As of October 16, 2019, 157 cases of African swine fever have been reported in China, with 1.192 million pigs killed, which has brought huge economic losses to the pig industry in China. African swine fever virus was the only known DNA arbovirus up to now, and was coated with an envelope. The genome size of ASFV was 170-190Kb, which can encode 150-200 types of proteins. Many genes of these proteins have been cloned and expressed. Among them, the protein encoded by B646L gene was a major capsid structural protein of African swine fever virus, accounting for 32% of the protein content of virus particles. African swine fever will not cause neutralization reaction, but studies have shown that the protein encoded by B646L gene has a conformational neutralizing epitope, which can induce the body to produce neutralizing antibody. But, unfortunately, this neutralizing antibody cannot protect animals from virus attack. However, the protein encoded by B646L gene was the main structural protein of ASFV capsid, which can induce specific humoral immune response. It was an immunogenic antigen with good antigen stability, which was an ideal antigen for serological diagnosis of ASFV. The most popular laboratory diagnostic methods for ASFV include .0 PCR, etiology and serology, among which ELISA and Western Blot were recommended by World Organization for Animal Health (OIE). Therefore, it was of great practical significance to develop more effective antibodies in serological diagnostic reagents for ASFV.

Contents of Invention .5 The purpose of this invention is to provide a monoclonal antibody which can compete with positive serum for binding to the African swine fever virus B646L antigen, including a competitive ELISA detection reagent containing the monoclonal antibody and a corresponding detection kit, as well as a method for detecting the African swine fever virus by using the detection reagent or the detection kit, so as to solve the disadvantages of the existing detection technology. Firstly, this invention provides an antigen polypeptide, the sequence of which is selected from one or more of the following: RRNIRFKPWFIPGVIN (SEQ ID NO:19); ALWIKLRFWFNENVNL (SEQ ID NO:20); FVTPEIHNLFVKRVRF (SEQ ID NO:21); RFIAGRPSRRNIRFKP (SEQ ID NO:22);

PGVINEISLTNNELYI (SEQ ID NO:23); QVTHTNNNHHDEKLMS (SEQ ID NO:24); SVSIPFGERFITIKLA (SEQ ID NO:25); MSALKWPIEYMFIGLK (SEQ ID NO:26); ERFITIKLASQKDLVN (SEQ ID NO:27); and SLTNNELYINNLFVTP (SEQ ID NO:28)o Secondly, this invention provides an antigen binding protein which can specifically compete with positive serum for binding to African swine fever virus B646L antigen, wherein the antigen binding protein comprises at least one .0 heavy chain variable region and at least one light chain variable region. The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:1 or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids to the amino acid sequence shown in SEQ ID NO:1; the light chain variable region has an amino acid sequence shown in .5 SEQ ID NO:2 or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids to the amino acid sequence shown in SEQ ID NO:2. where: The amino acid sequence shown in SEQ ID NO: lwas as follows: EVMLVESGGGLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKRLEWV ATEWSFGVPTSDSVKGRFIISRDNAKNTLYLQMSSLRSEDTAMYYCATE GPFMSQVGTLVTVSA; The amino acid sequence shown in SEQ ID NO: 2was as follows: DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKYSLLLVY NAKSLAEGVPSRFSGSGAGTQFSLKISSLQTEDFGSYYCQHHYGTPYTF GGGPKLEIK. The sequence of the antigen selected from one or more of the following is preferred: RRNIRFKPWFIPGVIN (SEQ ID NO:19); ALWIKLRFWFNENVNL (SEQ ID NO:20); FVTPEIHNLFVKRVRF (SEQ ID NO:21); RFIAGRPSRRNIRFKP (SEQ ID NO:22); PGVINEISLTNNELYI (SEQ ID NO:23); QVTHTNNNHHDEKLMS (SEQ ID NO:24); SVSIPFGERFITIKLA (SEQ ID NO:25); MSALKWPIEYMFIGLK (SEQ ID NO:26); .0 ERFITIKLASQKDLVN (SEQ ID NO:27); and SLTNNELYINNLFVTP (SEQ ID NO:28)o Thirdly, this invention provides an antibody or active fragment which can specifically compete for binding to the African classical swine fever virus B646L antigen, wherein the antibody or active fragment comprises at least one .5 heavy chain variable region and at least one light chain variable region. The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:1 or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids to the amino acid sequence shown in SEQ ID NO:1; The light chain variable region has an amino acid sequence shown in SEQ ID NO:2 or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids to the amino acid sequence shown in SEQ ID NO:2. The sequence of the antigen selected from one or more of the following is preferred: RRNIRFKPWFIPGVIN (SEQ ID NO:19); ALWIKLRFWFNENVNL (SEQ ID NO:20); FVTPEIHNLFVKRVRF (SEQ ID NO:21);

RFIAGRPSRRNIRFKP (SEQ ID NO:22); PGVINEISLTNNELYI (SEQ ID NO:23); QVTHTNNNHHDEKLMS (SEQ ID NO:24); SVSIPFGERFITIKLA (SEQ ID NO:25); MSALKWPIEYMFIGLK (SEQ ID NO:26); ERFITIKLASQKDLVN (SEQ ID NO:27); and SLTNNELYINNLFVTP (SEQ ID NO:28)o Preferably, the antibody or active fragment is a monoclonal antibody and/or a genetically engineered antibody. The genetically engineered antibody is selected .0 from one of the following: single chain antibody, single chain antibody fragment, chimeric monoclonal antibody, chimeric monoclonal antibody fragment, modified monoclonal antibody and modified monoclonal antibody fragment. More preferably, the antibody is a mouse monoclonal antibody T-2. .5 Fourthly, this invention provides a competitive ELISA detection kit for African swine fever virus, which comprises the antigen binding protein, antibody or active fragment that specifically competitively binds to the antigen of African swine fever virus B646L. Preferably, the antibody wis a mouse monoclonal antibody T-2. More preferably, the antigen binding protein, antibody or active fragment is an antigen binding protein, antibody or active fragment labeled with a marker. More preferably, the marker is selected from enzymes, fluorescent groups and chemiluminescent groups. In a preferable embodiment of the present invention, the competitive ELISA detection kit for African swine fever virus provided by the invention comprises a micro-plate coated with ASFV B646L antigen and an enzyme-labeled mouse monoclonal antibody T-2.

Preferably, the coating amount of the B646L antigen is 0.1-8 pg/mL. The enzyme-labeled mouse monoclonal antibody T-2 is diluted by a volume of 1: (2000-40000). Preferably, the B646L antigen is obtained by eukaryotic expression. Furthermore, the kit in this invention can also include sample diluent, blocking solution, washing solution, chromogenic solution, stop solution, ASFV standard positive serum and negative serum. Fifthly, the invention provides a preparation method of the African swine fever virus competitive ELISA detection kit, wherein the preparation of the .0 enzyme-labeled plate comprises: diluting antigen with carbonate buffer solution, coating the microporous enzyme-labeled plate, staying overnight; washing, adding 5% BSA, blocking, washing and drying. In a preferable embodiment of the present invention, the preparation method of the kit comprises the following steps: wherein the preparation of the .5 enzyme-labeled plate involves diluting 0.05 mol/L, pH9.6 carbonate buffer solution into 1 g/mL , coating the microporous enzyme-labeled plate with 100 pL per well, staying overnight under 4 °C; washing with washing liquid for 3 times, adding 200 pL of 5% BSA to each well, blocking at 37 °C for 1 hour, washing with washing liquid for 3 times, drying and vacuum packaging. Sixthly, the invention provides the application of the antigen binding protein, the antibody or the active fragment which specifically binds to the antigen of African swine fever virus B646L, or the competitive ELISA detection kit of African swine fever virus in detecting African swine fever virus in samples. Preferably, the sample is serum sample or blood sample. Seventhly, this invention provides a method for detecting African swine fever virus in a sample in vitro by using the antigen binding protein, the antibody or the active fragment which specifically binds to the antigen of African swine fever virus B646L, or the African swine fever virus competitive ELISA detection kit. Preferably, the sample is serum sample or blood sample. In a preferable embodiment of the present invention, the method comprises the following steps: (1) adding a sample to be tested into an enzyme-labeled micro-plate; (2) adding the enzyme-labeled antigen binding protein, the enzyme-labeled antibody or active fragment which has been diluted with diluent into the enzyme-labeled micro-plate and incubating; (3) after color development, .0 measuring OD 4 5 0mvalue and calculating PI value; and (4) when the PI of the detected serum is > 50%, it is positive; when the PI of the tested sample is < 40%, it is negative; when the tested sample is 40% < PI < 50%, it is suspicious. In a preferable embodiment of the present invention, the method for detecting the serum and blood samples of African swine fever virus by using the African .5 swine fever virus competitive ELISA detection kit comprises the following steps: Step (1) taking out the enzyme labeled plate and balancing it to room temperature; Step (2) diluting the sample to be detected twice with diluent, adding 50 pL per well to the enzyme-labeled micro-plate, diluting the enzyme-labeled monoclonal antibody T-2 with diluent 1:20000, and incubating at 37 °C for 30min after mixing evenly; Step (3) discarding the sample to be detected, washing it with washing solution for 5 times, patting it dry or swabbing off; Step (4) adding 100 pL TMB substrate chromogenic solution to each well, and developing color for 15 min; Step (5) adding 50 pL 2 mol/L H 2 SO4 to each well to stop color development, measuring the OD 4 5 0 n value and calculating the PI value. Step (6) result judgment: when the PI of the detected serum is > 50%, it is positive; when the PI of the detected serum is < 40%, it is negative; when the PI range of the detected serum is 40% < PI < 50%, it is suspicious. The beneficial effects of the invention: (1) The monoclonal antibody against ASFV B646L antigen provided by the invention has high potency and stable property, and can be prepared with mouse ascites and is suitable for mass production; (2) The potency of T-2 antibody after enzyme labeling is high, and the .0 competitive ELISA method for African swine fever established by this method has good repeatability, good specificity and high sensitivity. Compared with the similar imported commercial kit, it is easier to distinguish positive and negative results; (3) In the competitive ELISA detection kit for African swine fever virus .5 provided by the invention, the coated antigen (B646L antigen) can be obtained by eukaryotic expression, which is easy to produce, stable in effect, free of non-specific reaction, low in coating amount, and beneficial to mass production of the kit; (4) The competitive ELISA method for African swine fever virus provided by the invention has the advantages of rapidness, convenience, sensitivity, specificity and the like, and has more advantages in the detection of weak positive samples compared with the existing imported commercial kit, and has extensive application in the clinical detection of African swine fever. According to the technical scheme of the invention, some amino acids in the amino acid sequence can be conservatively substituted without changing the activity or function of the protein, as shown in Table 1 below:

Table 1

residue conservative residue conservative

substitution substitution

Ala Ser Leu Ile; Val

Arg Lys Lys Arg; Gln

Asn Gln; His Met Leu; Ile

Asp Glu Phe Met; Leu; Tyr

Gln Asn Ser Thr; Gly

Cys Ser Thr Ser; Val

Glu Asp Trp Tyr

Gly Pro Tyr Trp; Phe

His Asn; Gln Val Ile; Leu

Ile Leu; Val

In addition, because of the degeneracy of the base, the base of the polynucleotide sequence can be substituted without changing the activity or function of the polynucleotide sequence, see Table 2 below:

Table 2

The second position of codon T C A G TTT: Phe F TCT: Ser S TAT: Tyr Y TGT: Cys C T o T TTC: Phe F TCC: Ser S TAC: Tyr Y TGC: Cys C C oT TTC: LeT L TCA: Ser S TAA: Ter* TGA: Ter* A c TTC: LeT L TCG: Ser S TAG: Ter* TGG: Trp W G CTT: LeT L CCT: Pro P CAT: His H CGT: Arg R T

CTC: LeT L CCC: Pro P CAC: His H CGA: Arg R C CTA: LeT L CCA: Pro P CAA: Gln Q CGC: Arg R A CTG: LeT L CCG: Pro P CAG: Gln Q CGG: Arg R G ATT: Ile I ACT: Thr T AAT: Asn N AGT: Ser S T A ATC: Ile I ACC: Thr T AAC: AsnN AGC: Ser S C

ATA: Ile I ACA: Thr T AAA: Lys K AGA: Arg R A ATG: Met M ACG: Thr T AAG: Lys K AGG: Arg R G GTT: Val V GCT: Ala A GAT: Asp D GGT: Gly G T

GAC: Asp D GGC: Gly G C G GTC: Val V GCC: Ala A GTA: Val V GCA: Ala A GAA: Glu E GGA: Gly G A GTG: Val V GCG: Ala A GAG: Glu E GGG: Gly G G degenerate bases Normal base R A/G Y C/T M A/C K G/T S G/C W A/T H A/T/C B G/T/C V G/A/C D G/A/T N A/T/C/G

Description of Figures Figure 1 shows SDS-PAGE electrophoresis of purified B646L protein. Figure 2shows the Western blot results of monoclonal antibody T-2 and

inactivated virus.

Specific Mode for Carrying out the Invention The following was a further explanation to the invention, but the invention was

not limit to the description here in any way.

Examples

1. Materials

Fetal bovine serum, DMEM, HAT and HT were all products of Gibco, US; pcv3

positive serum, blue ear disease positive serum, seneca positive serum and

pseudorabies positive serum were kept by our laboratory. Fifty inactivated

African swine fever positive serum and 200 ASFV negative serum were donated

by the National Veterinary Research Institute, Poland and the National

Reference Laboratory of African Classical Swine Fever, Sardinia Institute of Animal Health, Italy. Fifteen inactivated swine positive serum obtained from the epidemic areas and 100 swine negative serum imported from the United States obtained from the non-epidemic areas were provided by the National Reference Laboratory of African Classical Swine Fever, Sardinia Institute of Animal Health, Italy. B646L standard antibody detection kit (Ingezim PPA Compac) was sourced from INGENASA, Spain. The myeloma cell SP2/0 was preserved in our laboratory, and Vero cell was also preserved in our laboratory. Trizol was sourced from Thermo, One step RNA RT-PCR Kit and rTaq were sourced from .0 TaKaRa, and other chemical reagents were analytically pure. HRP Conjugation Kit was sourced from abcam. pEASY©-T1 Cloning Kit was sourced from Beijing TransGen Biotech Co., Ltd. SPF 6~8 weeks BALB/c mice were sourced from Beijing Vital River Laboratory Animal Technology Co. Ltd; SPF piglets were sourced from Beijing .5 SPF Pig Breeding Management Center; The IgG subclass identification kit was the product of Sigma, US. 2. Acquisition and Purification of African Swine Fever B646L Antigen 2.1 Primer Design Based on the reference sequence of B646L gene published by GenBank, a pair of PCR primers was designed by Primer5.0 software, and EcoRI and BamHI restriction sites were introduced into the upstream and downstream primers, which were named as B646L-U and B646L-L. B646L-U: 5'-CGCGAATTCATGGCATCAGGAGGAGCTTTTTGTCTTATTGCTAA CGATG-3' (SEQ ID NO:3) B646L-L: 5'-ATAGGATTCCTGAAAGCTTATCTCTGCGTGGTGAGTAG-3' (SEQ

ID NO:4) The length of the amplified product was 1527bp, and the primers were synthesized by BGI Tech Solutions (Beijing Liuhe) Co., Limited. 2.2 Construction of Recombinant Expression Plasmid and Prokaryotic Expression and Purification of B646L Antigen The recombinant plasmid pET30a-B646L was obtained by the conventional method. The recombinant plasmid was transformed into E.coli BL21(DE3), and the single colony was picked and cultured in LB medium containing kanamycin (final concentration: 50 pg/mL) in shaking table at 37 °C and 225r/min until .0 logarithmic growth period (OD600=0.6~1.0), then IPTG was added, the temperature was changed to 16°C, and the expression of target protein was induced overnight. The expressed recombinant B646L antigen was purified by conventional purification method of Ni-NTA agarose resin. Finally, the target protein was eluted with 250 mmol/L imidazole, and the eluate was concentrated .5 with 10 ku ultrafiltration tube. The solution was changed and the protein was diluted with PBS, then 10 pL protein solution was taken for SDS-PAGE detection. SDS-PAGE electrophoresis results (fig. 1) showed that the purified target protein was 52kD in size, and the purified protein showed a single band, indicating that the protein purity was high. Meanwhile, the protein concentration determined by BCA protein determination kit was 2 mg/mL, and B646L antigen was subpackaged and stored under -80°C for later use. 3. Preparation and Sequencing of Monoclonal Antibody against B646L Antigen 3.1 Analysis and Synthesis of Polypeptides and Mouse Immunity On-line epitope analysis tools ABCpred Prediction https://webs.iiitd.edu.in/raghava/abcpred/ > Scratch ( http://scratch.proteomics.ics.uci.edu/ ) IEDB

(http://tools.immuneepitope.org/main/) was used to analyze the amino acid sequence of B646L gene, and 10 epitopes of B cell were screened out as follows: Table 3 Artificial Synthetic Peptides Sequence ID Sequence Location Score

1. RRNIRFKPWFIPGVIN (SEQ ID NO:19) 388-403 0.9

2. ALWIKLRFWFNENVNL (SEQ ID NO:20) 328-343 0.9

3. FVTPEIHNLFVKRVRF (SEQ ID NO:21) 418-433 0.88

4. RFIAGRPSRRNIRFKP (SEQ ID NO:22) 380-395 0.86

5. PGVINEISLTNNELYI (SEQ ID NO:23) 399-414 0.82

6. QVTHTNNNHHDEKLMS (SEQ ID NO:24) 442-457 0.81

7. SVSIPFGERFITIKLA (SEQ ID NO:25) 347-362 0.79

8. MSALKWPIEYMFIGLK (SEQ ID NO:26) 456-471 0.78

9. ERFITIKLASQKDLVN (SEQ ID NO:27) 354-369 0.70

10. SLTNNELYINNLFVTP (SEQ ID NO:28) 406-421 0.65

The 10 polypeptides mentioned above were artificially synthesized by Beijing Tsingke Biological Technology Co., Ltd., which was diluted to 1 mg/mL with PBS, and were mixed with a certain proportion which was established according to the score of polypeptide by software (high proportion for high scores, and low proportion for low scores) and used as the immunogen. Female BALB/c io mice aged 6-8 weeks were immunized according to the conventional methods and the spleen cells of the immunized mice were fused with mouse myeloma cells SP2/0 in good growth status according to the conventional method. 3.2 Establishment of Indirect ELISA Detection Method The optimal working concentration of coated antigen and antibody for indirect ELISA was determined by matrix titration. The purified B646L prokaryotic expression antigen was diluted to 1 mg/mL, then diluted with coating buffer

(0.05 mol/L, pH9.6 carbonate buffer) at 1:400, 1:600, 1:800, 1:1000, 1:1200 and 1:1400 respectively, and then the ELISA plate was coated with 100 pL antigen diluent per well, overnight under 4°C. The positive serum and negative serum of BALB/c mice were diluted at 1:100, 1:200, 1:400, 1:800, 1:1600 and 1:3200 respectively. At the same time, sp2/0 cell culture supernatant and blank control were prepared, and the optimal working concentration was the antigen dilution concentration with OD 4 5 0 value of about 1.0 and maximum P/N ratio. The results showed that the optimal dilution of antigen was 1:1000 (the final concentration of protein was Ipg/mL). .0 3.3 Establishment of Hybridoma Cell Line with Monoclonal Antibody (mAb) Against B646L Antigen With B646L antigen expressed in prokaryote as coating antigen, the specific antibody in supernatant of fusion cells was detected by established indirect ELISA. The cells with high antibody potency, monoclonal growth and good cell .5 status were selected for subclonal culture. When the antibody secretion was stable and the proportion of positive pores was more than 95%, the expended culture can be carried out. 3.4 Preparation and Potency Determination of Ascites Three monoclonal antibody cells were screened by indirect ELISA. One of the hybridoma cells T-2 with the highest potency was selected for expanded culture. After routine treatment, Balb/c mice treated with incomplete Freund's adjuvant were injected intraperitoneally to prepare ascites. The potency of ascites determined by indirect ELISA could reach 1:106. mAb in ascites was purified by

octanoic acid-ammonium sulfate method. Inactivated cytotoxicity was detected by SDS-PAGE, and T2 was used as primary antibody, and sheep anti-mouse HRP was used as secondary antibody. The reactivity of mAb was determined by Western blot. The results of Western blot (fig. 2) showed that there was reactivity between T-2 and ASFV. 3.5 Stability Identification of T-2 Monoclonal Antibody Indirect ELISA was used to measure the potency of the supernatants of the 1 t,0

2 0 th 3 th 4 0 th and 5 0 th generations of the screened T-2 hybridoma cells, so as to determine the stability of the hybridoma cells. The results showed that T-2 hybridoma cells could stably secrete monoclonal antibodies, and the potency remained basically at the same level. 3.6 Subtype Identification of Monoclonal Antibodies The subclass identification kit manufactured by Sigma was used to identify IgG .0 subclass of monoclonal antibody ascites, and the specific operation was carried out according to the instructions of the kit. By subtype identification, the subtype of T-2 monoclonal antibody was IgG2b. 3.7 Determination of Monoclonal Antibody Sequence 3.7.1 Extraction of RNA from Hybridoma Cells .5 When monolayer of the T-2 hybridoma cells was formed, the culture solution was discarded, then the cells were blown with 3 mL sterile PBS and counted. 1x106 cells were collected into a 1.5 mL centrifuge tube and centrifuged for 5 min at 800r/min. And then the RNA of hybridoma cells was extracted with RNA extraction kit according to the instructions. 3.7.2 RT-PCR Amplification and Identification of RNA Extracted from Hybridoma Cells Fourteen degenerate primers (including 4 for VH-F, 2 for VH-R, 6 for VL-F and 2 for VL-R) were designed, totaling 20 pairs. Table 4 Heavy and Light Chain RT-PCR Amplification Primer of Monoclonal Antibodies

Primers Sequence (5' to 3') mVL-F1 ATGGAGACAGACTCCTGCTAT (SEQ ID NO:5) mVL-F2 ATGGATTTTCAGGTGTTTTCAG (SEQ ID NO:6) mVL-F3 ATGRAGTCACAKACGGTCTTYRTA (SEQ ID NO:7) mVL-F4 ATGAGGKCCCHGCTYTYCTKGGR (SEQ ID NO:8) mVL-F5 ATGAAGTTGCCTGTGCTGTTG (SEQ ID NO:9) mVL-F6 ATGATGAGTCCTGCCTTCC (SEQIDNO:10) mVL-R1 ACTGGATGGTGGGAGGA (SEQ ID NO:11) VL-R2 CCCAAGCTTACTTGGGAAGATGGA (SEQ ID NO:12) mVH-F1 ATGGRATGSAGCTGMATSCTCTT (SEQID NO:13) mVH-F2 ATGRACTTCGGGYCTKGGTTTT (SEQ ID NO:14) mVH-F3 ATGGCTGTCTTGGGGCTCTTCT (SEQID NO:15) mVH-F4 ATGGRCAGTACHTYY (SEQ ID NO:16) mVH-R1 AYCTCCACACRCCAGTGGATAGAC (SEQID NO:17) VH-R2 CCCAAGCTTRCCARKGGATRA (SEQ ID NO:18)

Note: R=A/G, S=C/G, Y=C/T, M=C/A, K=T/G, H=A/T, D=G/T/A

With extracted RNA as template, primers were shown in table 4, reaction

system was 50 pL(5 pL for lOxOne Step RNA PCR Buffer , 10 pL for MgCl 2

(25 mM) , 10 pL for dNTP Mixture(10 mM) , 1 L for RNase Inhibitor (40 U/pL), 1 pl for AMV RTase XL (5 U/pL). 1I L of AMV-Optimized Taq (5 U/pL), 1.5 pL of F equivalent mixed primer, 1.5 pL of R equivalent mixed primer, 4 pL of template, 15 pL of ddH 2 0), reaction procedure (reverse

transcription at 50 °C for 30 min, pre-denaturation at 94 °C for 2 min,

denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72°C for

45 s, amplification for 35 cycles, and extension at 72°C for 10 min). Finally, 8

pL of the product was used for RT-PCR amplification by 1% agarose gel

electrophoresis. After identification, suitable primers were selected to amplify

VH and VL in large quantities (this step was repeated twice). The target band

was recovered by 2% agarose gel electrophoresis, and then VH and VL target

genes were recovered by gel recovery kit.

3.7.3 Linkage Transformation and Identification of VH and VL The recovered and purified VH and VL target genes were linked with

pEASY©-tl, and the reaction system was as follows: IpL pEASY©-T1 and 4 pL recovered and purified PCR products. The reaction was carried out at room temperature for 30 min. 50 pL of Trans 5a competent cells were melt on ice, and then ligation products were added in and mixed, following ice bathing for 30 min, heat shocking in 42°C water bath for 45s and ice bathing for 2-3 min. 600 pL LB liquid medium was added and resuscitated in shaker at 37 °C at a speed of 220 r/min for 60 min, 100 pL bacterial liquid was taken out and coated evenly on LB solid culture medium (AMP resistance), cultured in an incubator under 37 °C for 12-15 h, and observed the appearance of transformed single colony. 4-6 single colonies .0 in a 1.5 mL centrifuge tube (600 pL AMP resistant LB liquid medium) was chosen and cultured in a shaker at 37 °C for 220 r/min, and a control was set up. After culturing at 37 °C for 4 hours, 2.0 pL of bacteria solution was taken from each centrifuged tube for PCR identification. The reaction system was 20 pL(0.2 pL of rTaq enzyme (5 U/pL), 2.0 pL of 1OxPCR buffer, 2.0 pL of dNTP .5 (2.5 mM), 1.0 pL of M13-F(10pM) , 1.0 pL of M13-R(10pM) , 2.0 pL of bacteria solution and 9.8 pL ddH20), reaction procedure (pre-denaturation at 95 °C for 4 min, denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 45 s, amplification for 32 cycles, and another round of extension at 72 °C for 10 min). Finally, 8 pL of the product was used to observe the PCR amplification results by 1% agarose gel electrophoresis, and the positive clone solution was selected and submitted for sequencing. Three confirmed positive clones of T-2 monoclonal antibody light and heavy chain gene were selected as sequencing samples, and the nucleotide sequences of antibody variable regions were obtained. The results were compared by DNAStar and IgBLAST software. The results showed that the three light chain variable region genes were all 321bp long and encoded with 107 amino acids. The gene sequences of the three heavy chain variable regions were all 336bp long and encode with 112 amino acids. The variable region of the heavy chain has the amino acid sequence as shown in the following SEQ ID NO:1: EVMLVESGGGLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKRLEWV ATEWSFGVPTSDSVKGRFIISRDNAKNTLYLQMSSLRSEDTAMYYCATE GPFMSQVGTLVTVSA. The variable region of the light chain has the amino acid sequence as shown in the following SEQ ID NO:2: DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKYSLLLVY NAKSLAEGVPSRFSGSGAGTQFSLKISSLQTEDFGSYYCQHHYGTPYTF .0 GGGPKLEIK. 4. Establishment of Competitive ELISA for ASFV B646L Monoclonal Antibody 4.1 Reagent Preparation (1) Coating solution: 0.05 mol/L carbonate buffer solution with pH 9.6. .5 (2) Washing solution: pH7.4, 0.1M PBS, 0.05% Tween-20. (3) Blocking solution: preparing 5% BSA solution with washing solution. (4) Diluent: used as the blocking solution. (5) TMB: solution A: 0.02% H 2 0 2 , diluted with 0.1M citric acid -0.2M disodium hydrogen phosphate solution with pH5.0; solution B: 0.4%o TMB-HCl, dissolved with 50mM sodium citrate solution with pH2.8. 50 pL solution A and 50 pL solution B were taken respectively, then mixed and protected from light for later use. (Commercial TMB chromogenic solution can also be used) (6) Stop solution: 2M H 2 SO4 solution.

4.2 Preparation of ASFV B646L Coated Antigen

4.2.1 Gene Synthesis and Bacmid Extraction B646L and B602L gene sequences (GenBank: MK333180.1) were synthesized by Beijing Tsingke Biology Technology Co., Ltd. and constructed into pCDN4.1 vector. 4.2.2 Co-transfection of Two Plasmids of B646L and B602L Genes into 293F Cells 1) 293F cells with good growth status were prepared in advance and inoculated in 6-well plate, and 2 ml complete culture medium without antibiotics was added to ensure that the cells confluent up to 70~80% during .0 transfection. 2) 10 pg DNA (1: 1 for plasmids of B646L and B602L) was diluted in 500 pL serum-free and antibiotic-free culture medium and mixed gently; 20 pL freestyle was diluted in 500 pL serum-free and antibiotic-free culture medium and mixed gently, then incubated at room temperature for 5 min. After 5 .5 minutes, the above two mixed solutions were mixed gently and incubated at room temperature for 20 minutes. The total time should not exceed 25 min. 3) The culture solution in the culture dishes was absorbed and discarded, and 700 pL serum-free and antibiotic-free culture solution was added to each dish; the complex was added into culture dishes, then the culture dishes were shaken back and forth to be evenly distributed and incubated in an incubator at 37 °C for 4 ~ 6 hours, then the culture solution was discarded, and 10 mL fresh 293F cell culture solution containing 10%FBS was added and cultured continuously for 72 hours. 4.2.3 Collection of the Expressed Protein 293F cells expressing protein were collected, added with 20 mM hepes buffer, pH7.4, 300 mM NaCl solution and PMSF protease inhibitor, and sonicated on ice for 3 min. Then the samples were centrifuged at 16000rpm for 20min at 4°C and the supernatant was combined with his-NTA beads for 1 h. The hybrid protein was eluted with 20mM imidazole buffer (20mM Tris-HCl, 300mM NaCl, 20mM imidazole, pH7.5), and the target protein was eluted with 200mM imidazole (20mM Tris-HCl, 300mM NaCl, 200mM imidazole, pH7.5). The target protein was concentrated with a 1OKD ultrafiltration concentration tube, and a proper amount of samples was taken for SDS-PAGE detection. After the purity meets the requirements, the protein concentration was measured, which was the antigen of this kit, and the protein was stored at -80 °C after sub-packaging for later use. .0 4.3 Preparation of Standard ASFV Positive Serum and Standard ASFV Negative Serum In order to reduce the errors between different operators and different detection batches in the actual detection process with ELISA kit, standard ASFV antibody positive control serum and standard ASFV antibody negative control serum .5 were prepared for optimizing the detection method and determining the result adjudgment standard. 4.3.1 Preparation of Standard Positive Serum Six-week-old SPF piglets were selected, and disinfected the skin of injection site with iodine. In the first immunization, 5 ml of Freund's complete adjuvant (FCA) was absorbed by syringe and emulsified with the same amount of purified eukaryotic expression B646L antigen (2 mg/mL), and piglets were immunized by intramuscular injection. After an interval of 10-14 days, the piglets were immunized for the second time. After absorbing 8 ml Freund's incomplete adjuvant and emulsified with the purified eukaryotic expression B646L antigen, the piglets were immunized by intramuscular injection in the same way. After an interval of 7-10 days, blood was collected from the anterior vena cava, and the serum potency was detected by ELISA. When the antibody potency reached more than 1:100, blood was collected from the heart, and the separated serum was positive serum, which was preserved by adding 1/10,000 thiomersal, packed in 0.5 ml aliquots and stored at -20 °C for later use. 4.3.2 Preparation of Standard Negative Serum Blood was taken from the heart of SPF pigs that have passed the inspection, and the separated serum was negative serum, which was preserved by adding 1/10000 thiomersal, packed in sterile tubes with 0.5 ml aliquots and stored at -20 °C for later use. 4.4 Preparation of Enzyme-labeled Antibody and Determination of Optimal .0 Coating Concentration of Antigen and Optimal Dilution of Sample to Be Detected The purified B646L monoclonal antibody T-2 was labeled with HRP using the HRP conjugation Kit manufactured by abcam, and the required enzyme-labeled antibody was obtained. According to chessboard titration, B646L eukaryotic .5 expression antigen was coated with 100 pL antigen coating solution per well at the concentration of 0.1 pg/mL, 0.5 pg/mL, 1 pg/mL, 2 pg/mL, 4 pg/mL and 8 pg/mL, respectively, and stayed overnight at 4 °C. The coating solution was dumped the next day, washed with washing solution for three times, and patted dry, and 200 pL 5% BSA prepared with washing solution was added to each well. The standard negative and positive serum was diluted from 0, 1: 2 to 1:32 after washing for 3 times. They were added into 96-well enzyme-labeled micro-plates from top to bottom, with 50 pL per well. At the same time, the same volume of 1:10000 diluted enzyme-labeled antibody was added to each well. After shaking and mixing, they were incubated at 37 °C for 30 min, washed with washing solution for 5 times and patted dry. 100 pL TMB chromogenic solution was added to each well to develop color at room temperature for 15 min. The reaction was stopped by adding 50 pL stop solution to each well, and was read using the microplate reader at 450 nm. According to the P/N value, the best antigen coating concentration and the best enzyme-labeled antibody dilution were selected. The results showed that when the concentration of antigen coating was 1I g/nL and the dilution of serum was 1:2, the P/N value reached the maximum which was above 10, so the optimal concentration of antigen coating was 1 g/mL and the optimal dilution of sample was 1:2. 4.5 Optimization of Optimal Dilution of Enzyme-labeled Antibody The method was the same as section 4.4, and the enzyme-labeled antibody was .0 diluted 1:2000, 1:5000, 1:10000, 1:20000 and 1:40000. According to the best antigen coating concentration and the best sample dilution determined by standard negative and positive serum, the optimization experiment of the best enzyme-labeled antibody dilution was carried out under the same other conditions. After the reaction, the absorbance value at 450nm was read by the .5 microplate reader, and the P/N value was calculated. According to the P/N value, the best dilution of enzyme-labeled antibody was determined to be 1:20000. 4.6 Determination of the Best Blocking Solution Method applied was the same as above, 10% bovine serum, 5% BSA, 5% skimmed milk powder and 1% gelatin were used as blocking solutions at 37 °C for 1 h, and other conditions were tested according to the determined optimal conditions. The serum samples to be tested were detected, and the inhibition rate (PI) of the serum to be tested from each known background was calculated. According to PI value, the best blocking solution was determined as 5% BSA at 37 °C for 1 h. OD4so .m of standard negative serum-OD 45 0 . of sample serum Inhibition rate (PI)= x100% OD4 5 0 umof standard negative serum-OD5o of standard positive serum

4.7 Determination of the Best Color Developing Time According to the best reaction conditions determined above, after adding TMB chromogenic solution, the color of the samples was developed at room temperature for 5 min, 10 min, 15 min and 20 min, respectively. Keeping other conditions unchanged, all kinds of serum to be detected were detected. According to the inhibition rate (PI) of serum with known background, the best action time of TMB was determined to be 15 min under room temperature. 4.8 Determination of Criteria for Determining Positive and Negative Serum by Competitive ELISA To determine the boundary of positive and negative serum in c-ELISA, the serum of known positive serum (from the National Reference Laboratory of .0 African Classical Swine Fever, Sardinia Institute of Animal Health, Italy and the National Veterinary Research Institute, Poland) and the serum from non-epidemic swines which were used as known negative serum were used, and the established monoclonal antibody c-ELISA method was employed to detect 50 known positive serum and 200 known negative serum for three times, .5 respectively, in order to calculate the inhibition rate of each serum sample. The results showed that the PI% values of 50 positive serum were all over 50%, and those of 200 negative serum were all less than 40%. Therefore, the criteria for determining the positive and negative serum samples of monoclonal antibody c-ELISA established in this study was as follows: when the serum PI>50%, it was positive; when serum PI<40%, it was negative; when the range of serum PI was 40% < PI < 50%, it was suspicious. 4.9 Repeatability Test According to the coefficient of variation inter- and intra-batch, the stability of c-ELISA was evaluated. Intra-batch error: 10 serum samples were detected with the same batch of coated ELISA plates, and each serum was prepared with 3 replicates, and the coefficient of variation was calculated to indicate the intra-batch error. Inter-batch error: the ELISA plates were coated at 3 different times, and 10 serum samples were detected at the same time. Each sample was provided with two replicated wells, and the coefficient of variation inter-batch was calculated to indicate the inter-batch error. The results of intra-batch repeated experiments and inter-batch repeated experiments showed that the intra-batch coefficient of variation and inter-batch coefficient of variation were both less than 10%, thus we drew the conclusion that the monoclonal antibody c-ELISA method of this invention had better stability and repeatability. 4.10 Specific Evaluation of Monoclonal Antibody c-ELISA .0 According to the c-ELISA procedure determined above, ASFV standard positive serum, pcv3 positive serum, blue ear disease positive serum, seneca positive serum and pseudorabies positive serum were detected respectively. The inhibition rate was calculated according to the color development results and formula, and the specificity of monoclonal antibody c-ELISA was evaluated by .5 the inhibition rate. Test results showed that the PI% values of pcv3 positive serum, blue ear disease positive serum, seneca positive serum and pseudorabies positive serum were all lower than 50%, and the PI% values of ASFV standard positive serum and ASFV positive serum were all higher than 50% (table 5). Therefore, we drew the conclusion that the competitive ELISA method of T-2 monoclonal antibody could distinguish several common diseases of pigs from ASFV. Table 5 Specific Detection Results of Monoclonal Antibody c-ELISA

Serum Types Inhibition Rate (PI%) ASFV standard positive serum 100 pcv3 positive serum 9.32

Blue ear disease positive serum 9.69 Seneca positive serum 10.56

Pseudorabies positive serum 9.23 ASFV standard negative serum 0

4.11 Detection of Clinical Samples Using the optimized c-ELISA reaction conditions, 15 inactivated swine positive serum obtained from the epidemic areas and 100 swine negative serum imported from the United States obtained from the non-epidemic areas were detected, and the results were compared with the standard c-ELISA kit. The detection results of the two kits were shown in Table 6 and Table 7. The detection results of negative samples were consistent, but the weak positive samples could only be detected by the kit developed by our institute and not by the imported commercial kit. Moreover, the detection value of the kit in this o study was more obvious, and it had more advantages than the commercial kit in detecting weak positive samples. Table 6 Results of Positive Samples by C-ELISA and Imported Kits

Sequence Sample # c-ELISA The kit used in our invention

number for OD450nm X% Adjudg OD450nm P1% Adjudg the samples ment ment

1. S45 0.133 90.60606 + 0.102 98.82698 +

2. M6 0.102 100 + 0.096 100.5865 +

3. D4 0.11 97.57576 + 0.097 100.2933 +

4. D9 0.101 100.303 + 0.089 102.6393 +

5. S33 0.113 96.66667 + 0.099 99.70674 +

6. S50 0.156 83.63636 + 0.115 95.01466 +

7. 90 0.213 66.36364 + 0.126 91.78886 +

8. 38 0.307 37.87879 - 0.135 89.14956

+ 9. 274 0.198 70.90909 + 0.124 92.37537

+ 10. 268 0.205 68.78788 + 0.112 95.89443

+ 11. 339 0.113 96.66667 + 0.093 101.4663

+ 12. F47 0.119 94.84848 + 0.101 99.12023

+ 13. D91 0.117 95.45455 + 0.098 100

+ 14. 668 0.114 96.36364 + 0.095 100.8798

+ 15. 585 0.115 96.06061 + 0.087 103.2258

+ 16. Positive 0.102 / + 0.098 /

+ 17. Negative 0.432 / - 0.439 /

Table 7 Test Results of the Negative Samples by c-ELISA and Imported Kit

Methodology The kit used in our c-ELISA Sample Status invention

100 negative samples 100 100

Coincidence rate of test results 100%

4.12 Preparation of Enzyme Labeling Plate B646L eukaryotic expression antigen was diluted with the coating solution according to the best coating concentration (1 g/mL), added into a 96-well enzyme-labeled microplate with the amount of 100 pL/well and kept overnight under 4 °C. The solution in the wells was poured out the next day, washed with washing solution for 3 times and finally patted dry. Then 200 pL of blocking solution (5% BSA) was added to each well, sealed at 37 °C for 1 hour, washed with washing solution for 3 time, and then dried and vacuum packaged. 4.13 Operation Steps of ASFVc-ELISA Detection Kit 4.13.1 The sample to be detected was diluted with sample diluent 1:2, and 50 pL diluted serum to be detected was added to each well, then 50 pL 1:20000 diluted enzyme-labeled monoclonal antibody was added. Standard negative and positive control was set up and incubated at 37 °C for 30 min, and 100 pL TMB substrate chromogenic solution was added to each well after washing five times to develop color at room temperature for 15 min. The color developing reaction was stopped by adding 2 mol/L H 2 SO4 50pL/well. OD 45 0 mvalue was measured o and serum inhibition rate was calculated according to the formula. 4.13.2 Result adjudgment: when the PI of the detected serum was > 50%, it was positive; when the PI of the detected serum was < 40%, it was negative; when the PI range of the detected serum was 40% < PI < 50%, it was suspicious.

Claims

Claims 1. An antigen binding protein which can specifically compete with positive serum for binding to African swine fever virus B646L antigen, wherein the antigen binding protein comprises at least one heavy chain variable region and at least one light chain variable region; the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:1 or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids to the amino acid sequence shown in SEQ ID NO:1; the light chain variable region has an amino acid sequence shown in SEQ ID NO:2 or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids .0 to the amino acid sequence shown in SEQ ID NO:2.
2. An antibody or active fragment which can specifically compete for binding to the African classical swine fever virus B646L antigen, wherein the antibody or active fragment comprises at least one heavy chain variable region and at least one light chain variable region; the heavy chain variable region has .5 the amino acid sequence shown in SEQ ID NO:1 or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids to the amino acid sequence shown in SEQ ID NO:1; the light chain variable region has an amino acid sequence shown in SEQ ID NO:2 or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids to the amino acid sequence shown in SEQ ID NO:2.
3. The antibody or active fragment according to claim 2, wherein the antibody or active fragment is a monoclonal antibody and/or a genetically engineered antibody; the genetically engineered antibody is selected from one of the following: single chain antibody, single chain antibody fragment, chimeric monoclonal antibody, chimeric monoclonal antibody fragment, modified monoclonal antibody and modified monoclonal antibody fragment; preferably, the antibody is mouse monoclonal antibody T-2.
4. A competitive ELISA detection kit for African swine fever virus, wherein the kit comprises the antigen binding protein mentioned in claim 1, or the antibody or active fragment mentioned in claim 2 or 3.
5. The kit according to claim 4, wherein the antibody is mouse monoclonal antibody T-2.
6. The kit according to claim 4 or 5, wherein the antigen binding protein, antibody or active fragment is an antigen binding protein, antibody or active fragment labeled with a marker; preferably, the marker is selected from .0 enzymes, fluorescent groups and chemiluminescent groups.
7. The kit according to claim 4 or 5, wherein the kit comprises a micro-plate coated with ASFV B646L antigen and an enzyme-labeled mouse monoclonal antibody T-2; preferably, the coating amount of the B646L antigen is 0.1-8 pg/mL, the .5 enzyme-labeled mouse monoclonal antibody T-2 is diluted by a volume of 1: (2000-40000); preferably, the B646L antigen is obtained by eukaryotic expression.
8. Application of the antigen binding protein mentioned in claim 1, the antibody or active fragment mentioned in claim 2 or 3, or any kit mentioned in claims 4 to 7 in detecting African classical swine fever virus in a sample; preferably, the sample is a serum sample or a blood sample.
9. A method for detecting African classical swine fever virus in a sample in vitro by using the antigen binding protein mentioned in claim 1, the antibody or active fragment mentioned in claim 2 or 3, or any kit mentioned in claims 4 to 7; preferably, the sample is a serum sample or a blood sample.
10. The method according to claim 9, wherein the method comprises the following steps:

(1) adding a sample to be tested into an enzyme-labeled micro-plate; (2) adding the enzyme-labeled antigen binding protein mentioned in claim 1, or the enzyme-labeled antibody or active fragment mentioned in claim 2 or 3 which has been diluted with diluent into the enzyme-labeled micro-plate and incubating; (3) after color development, measuring OD 45 0 nm value and calculating PI value; and (4) when the PI of the detected serum is > 50%, it is positive; when the PI of the detected serum is < 40%, it is negative; when the PI range of the detected serum is 40% < PI < 50%, it is suspicious.