Competitive ELISA kit for detecting antibodies against African swine fever virus p30 protein

Abstract

The present invention belongs to the field of biotechnology and relates to a competitive ELISA kit for detecting antibodies to the p30 protein of African swine fever virus (ASFV). The kit comprises: an enzyme label plate coated with ASFV p30 protein, an HRP-labeled monoclonal antibody to ASFV p30 protein, a positive serum control, and a negative serum control. The heavy chain constant region of the monoclonal antibody to the ASFV p30 protein is of the IgM type, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4. The kit of the present invention has no cross-reaction with positive sera of common swine disease-related viruses, and the intra-batch and inter-batch repeat coefficients of variation are less than 10%. The conformity rate of the blocking ELISA kit of the present invention with that of the foreign commercial kit is 96.69%, and the Kappa value is 0.93, and the two are highly consistent.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a competitive ELISA kit for detecting antibodies against p30 protein of African swine fever virus. Background Art

[0002] African swine fever (ASF) is caused by African swine fever virus (ASFV), an ancient disease of pigs. At present, the prevention and control of African swine fever mainly adopts continuous monitoring and regionalized prevention and control management to prevent the invasion of the virus.

[0003] Serological antibody detection is an important means of diagnosing ASF. In endemic countries or countries without African swine fever, ELISA is often used to diagnose ASFV infection and assess the prevalence of the disease. The European Union has approved three antibody detection ELISA kits, including Ingenasa, IDvet and Svanovir, with an average accuracy of about 80%. At present, many ELISA serological tests are based on structural proteins, among which p30 is the most commonly used diagnostic antigen. The p30 protein is encoded by the CP204L gene and has a molecular weight of about 23.6kDa. The p30 protein can be detected 2 to 4 hours after ASFV infects cells and is an early expressed protein. Therefore, the p30 protein can be used as a detection target for diagnosing early ASFV infection.

[0004] After ASFV infects the body, antibodies can be produced within 7 to 10 days. The first thing that appears after the virus invades the body is the immunoglobulin IgM antibody, which takes about 5 to 7 days to produce, and then the IgG antibody appears in 10 to 15 days. Summary of the invention

[0005] The present invention screened out a monoclonal antibody specific to the ASFV p30 protein, and based on this, developed a competitive ELISA kit for detecting ASFV p30 protein antibodies. The kit takes less time than indirect and blocking ELISAs and has good specificity and sensitivity. In addition, the kit can detect antibodies as early as the 7th day after ASFV infection, which is of great significance for the early prevention and control of ASF.

[0006] The first object of the present invention is to provide a monoclonal antibody specific for ASFV p30 protein.

[0007] The second object of the present invention is to provide a competitive ELISA detection kit for ASFV p30 protein antibodies, which is easy to operate, short in time, has good specificity and high sensitivity, and can be used for early detection of ASFV infection.

[0008] The third object of the present invention is to provide an application of the above-described kit in the preparation of a reagent for detecting ASFV antibodies in pig serum.

[0009] In order to achieve the purpose of the present invention, the present invention provides the following technical solutions:

[0010] The present invention uses a prokaryotic expression system to obtain a soluble ASFV p30 protein with immunogenicity and reactivity, and uses it as an immunogen to immunize mice, and uses cell fusion and subclone screening technology to obtain a monoclonal antibody specific to the ASFV p30 protein, which is named 2A6. The present invention uses nested PCR amplification technology to obtain the variable region gene sequence of antibody 2A6, which can be subsequently obtained by genetic engineering or protein engineering methods.

[0011] The present invention is also based on the monoclonal antibody specific to ASFV p30 protein to prepare a competitive ELISA kit for detecting ASFV p30 protein antibodies. The kit includes: an ELISA plate coated with ASFV p30 protein, an HRP-labeled ASFV p30 protein monoclonal antibody (i.e., antibody 2A6), a positive control serum, and a negative control serum.

[0012] The preparation method of ASFV p30 protein is as follows: using ASFV p30 protein (Genbank accession number: MK128995.1) as a reference, designing specific primers, obtaining p30 gene by PCR amplification, connecting it with the prokaryotic expression vector pColdI vector, constructing recombinant plasmid pColdI-p30, transforming Escherichia coli BL21 (DE3), obtaining recombinant p30 protein by IPTG induction expression, and then using nickel column purification to obtain purified recombinant p30 protein. The amino acid sequence of the ASFV p30 protein is shown in SEQ ID NO: 1, and the DNA sequence encoding the ASFV p30 protein is shown in SEQ ID NO: 2.

[0013] SEQ ID NO: 1:

[0014] MKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIYGTPLKEEEKEVVRLMVIKLLKKK*;

[0015] SEQ ID NO:2:

[0016] .

[0017] Furthermore, the method for preparing the ASFV p30 protein monoclonal antibody comprises the following steps:

[0018] Purified recombinant p30 protein was used as an immunogen to immunize Balb / c mice at a dose of 30 μg / mouse. For the first immunization, the recombinant p30 protein was mixed with equal volumes of Freund's complete adjuvant and then emulsified, and multiple subcutaneous injections were performed on the back and abdomen. Booster immunizations were performed every 2 weeks, for a total of four immunizations. Booster immunizations were performed by mixing equal volumes of recombinant p30 protein and Freund's incomplete adjuvant and emulsifying them, and the immunization method was the same as the first immunization. After the fourth immunization, mouse spleen cells were fused with myeloma cells SP2 / 0 cells to prepare hybridoma cells, and the cell supernatants were verified by indirect ELISA and indirect immunofluorescence assay (IFA), and positive clones were screened. After three subclones, the hybridoma cells were injected into mice for the preparation of ascites, and then ASFVp30 protein monoclonal antibodies were obtained.

[0019] The amino acid sequence of the heavy chain variable region of the ASFV p30 protein monoclonal antibody is shown in SEQ ID NO: 3; the amino acid sequence of the light chain variable region of the ASFV p30 protein monoclonal antibody is shown in SEQ ID NO: 4. Among them, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain and light chain variable regions of the monoclonal antibody are as follows:

[0020] —— CDR1 CDR2 CDR3 Heavy chain VH GFSLTSYG IWSGGSTD AFYAMDY Light chain VL ESVDNYGISF AAS QQSKEVPWT

[0021] The DNA sequence encoding the heavy chain variable region of the ASFV p30 protein monoclonal antibody is shown in SEQ ID NO:5; the DNA sequence encoding the light chain variable region of the ASFV p30 protein monoclonal antibody is shown in SEQ ID NO:6.

[0022] SEQ ID NO:3:

[0023] QVQMKESGPGLVQPSQSLSITCTVSGFSLTSYGVHWVRQSPGKGLEWLGVIWSGGSTDYNAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYCAFYAMDYWGQGTSVTV;

[0024] SEQ ID NO:4:

[0025] TQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPWTFGGGTKLELK;

[0026] SEQ ID NO:5:

[0027] CAAGTGCAGATGAAGGAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTCATTAACTAGCTATGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATATGGAGTGGTGGAAGC ACAGACTATAATGCAGCTTTCATATCCAGACTGAGCATCAGCAAGGACAATTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAAGCTAATGACACAGCCATATATTACTGTGCGTTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTC;

[0028] SEQ ID NO:6:

[0029] ACCCAGTCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAGAGCCAGCGAAAGTGTTGATAATTATGGCATTAGTTTTATGAACTGGTTCCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAACCA AGGATCCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTATGGAGGAGGATGATACTGCAATGTATTTCTGTCAGCAAAGTAAGGAGGTTCCGTGGACGTTCGGTGGAGGGACCAAGCTGGAGCTGAAA.

[0030] The positive control serum in the competitive ELISA kit for detecting ASFV p30 protein antibodies is pig serum collected after ASFV artificial immunization; the negative control serum is pig serum without ASFV pathogens.

[0031] Furthermore, the competitive ELISA kit for detecting ASFV p30 protein antibodies also includes a coating solution, a blocking solution, a sample diluent, an enzyme-labeled antibody diluent, a washing solution, a color developing solution and a stop solution.

[0032] The coating solution is 0.05M carbonate buffer (1.5g Na2CO3, 2.92g NaHCO3 added to deionized water, pH adjusted to 9.6, and then fixed to 1L).

[0033] The blocking solution was a PBST solution containing 5% skim milk, 5% BSA or 2% trehalose. 5% skim milk, 5% BSA and 2% trehalose respectively indicated that 5 g skim milk, 5 g BSA and 2 g trehalose were added into every 100 mL PBST solution.

[0034] The sample diluent was 1% BSA in 0.01M pH 7.4 PBS solution (0.2 g KH2PO4, 2.9 g Na2HPO4·12H2O, 8 g NaCl, 0.2 g KCl dissolved in 800 mL ultrapure water, adjusted to pH 7.4 with NaOH, and diluted to 1 L with water).

[0035] The enzyme-labeled antibody diluent is a 0.01M pH 7.4 PBS solution containing 4% sucrose, 0.05% Tween-20, and 8% glycerol; the washing solution is 20 times concentrated PBST (5.4g KH2PO4, 28.4g Na2HPO4·12H2O, 160g NaCl, 4g KCl, 10mL Tween-20 are added to deionized water, the pH is adjusted to 7.4, and the volume is fixed to 1L).

[0036] The color developing solution is TMB, and the stop solution is 0.5M H2SO4.

[0037] The use of the above-mentioned competitive ELISA kit for detecting ASFV p30 protein antibodies includes the following steps:

[0038] (1) Coating: The purified ASFV p30 protein was diluted with coating solution and coated on the ELISA plate at 4°C overnight. The liquid in the plate was discarded, washed 4 times, and patted dry with absorbent paper;

[0039] (2) Blocking: Add blocking solution to block the ELISA plate, discard the liquid in the plate, wash 4 times, and pat dry with absorbent paper;

[0040] (3) Sample addition: Add the test serum diluted with sample diluent and the enzyme-labeled antibody (HRP-labeled ASFV p30 protein monoclonal antibody diluted with enzyme-labeled antibody diluent) mixed solution for reaction, and set up negative control, positive control and blank control wells. After discarding the liquid in the plate, wash 4 times and pat dry with absorbent paper;

[0041] (4) Color development: Add TMB substrate and protect from light;

[0042] (5) Termination: Add 0.5 M H2SO4 to terminate the reaction;

[0043] (6) Reading: OD measured by microplate reader 450 ;

[0044] (7) Calculate the blocking rate: PI = (negative control OD value - serum OD value to be tested) / negative control OD value × 100%. When the PI of the test sample is ≥46.30%, it is judged as positive, and PI <46.30% is judged as serum antibody negative.

[0045] As a preferred embodiment, the coating concentration of the ASFV p30 protein is 0.25 μg / mL.

[0046] As a preferred solution, the dilution ratio of the serum sample to be tested is 1:4.

[0047] As a preferred embodiment, the dilution multiple of the HRP-labeled ASFV p30 protein monoclonal antibody is 1:4000.

[0048] The beneficial effects of the present invention are:

[0049] The present invention uses purified ASFV p30 protein as an antigen and ASFV p30 protein monoclonal antibody as a detection antibody, and through a series of reaction conditions and reagent optimization, establishes a competitive ELISA kit and a detection method for detecting ASFV p30 protein antibodies. The competitive ELISA kit established by the present invention has good specificity, sensitivity, intra-batch and inter-batch repeatability and stability. The compliance rate with imported kits is as high as 96.69%, the Kappa value is 0.93, and the earliest antibodies can be detected 7 days after ASFV infection, indicating that the blocking ELISA kit established by the present invention can be used for early detection of ASFV p30 protein antibodies, and has broad market prospects and good economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is the expression and purification result of p30 recombinant protein (rp30);

[0051] AM: protein marker; 1: before rp30 induction; 2-3: after rp30 induction; 4: supernatant of rp30 lysis; 5: precipitate after rp30 lysis;

[0052] BM: protein marker; 1: rp30 purified protein;

[0053] Figure 2 The reactivity of the monoclonal antibodies was verified by indirect immunofluorescence assay.

[0054] A. 2A6 hybridoma cell supernatant; B. SP2 / 0 cell supernatant.

[0055] Figure 3 To identify the reactivity of monoclonal antibodies for Western blot;

[0056] M: protein marker; WT: ASFV-infected MA104 cells; Mock: MA104 cell control.

[0057] Figure 4 This is the result of monoclonal antibody subclass identification.

[0058] Figure 5 The results are after monoclonal antibody purification.

[0059] M: protein marker; 1: 2A6 after purification.

[0060] Figure 6 This is the result of PCR amplification of the variable region of 2A6 monoclonal antibody;

[0061] A. PCR amplification results of heavy chain variable region. M: DL2000 marker; VH: PCR amplification product of 2A6 heavy chain variable region.

[0062] B. PCR amplification results of the light chain K variable region. M: DL2000 marker; VL: PCR amplification product of the 2A6 light chain variable region.

[0063] Figure 7 This is the specific detection result of the ASFV p30 protein antibody competition ELISA method.

[0064] Figure 8 To detect serum antibody levels in artificially infected animals. DETAILED DESCRIPTION

[0065] The present invention is described in more detail below through specific implementation modes to facilitate understanding of the technical solution of the present invention, but is not intended to limit the protection scope of the present invention.

[0066] Example 1 Construction, expression and purification of ASFV p30 protein recombinant plasmid

[0067] 1.1 Construction of pColdI-p30 recombinant plasmid and induced expression and purification of p30 protein

[0068] With reference to ASFV p30 protein (Genbank accession number: MK128995.1), specific primers were designed, upstream primer P1: 5′-CGCGGATCCATGAAAATGGAGGTCATCTT-3′, downstream primer P2: 5′-ACGCGTCGACTTTTTTTTTAAAAGTTTAATAACC-3′, and BamHI and SalI restriction sites were introduced at the 5′ ends of the upstream and downstream primers. After primer synthesis, PCR amplification was performed using ASFVDNA as a template. The p30 gene PCR gel recovery product and the pColdI vector were double-digested with BamHI and XhoI, respectively, and then connected and transformed into DH5α competent cells. The constructed recombinant plasmid was named pColdI-p30.

[0069] The recombinant plasmid pColdI-p30 was transformed into E. coli BL21 (DE3), and a single colony was picked and placed in ampicillin-resistant LB medium. 600 When the concentration was about 0.8, IPTG with a final concentration of 1 mmol / L was added to induce expression. 5 hours after induction, the precipitate was collected by centrifugation at 12000 rpm for 2 minutes. The precipitate was resuspended with an appropriate amount of PBS and then ultrasonically treated for 5 minutes (ultrasonication for 3 seconds, stop for 3 seconds). After ultrasonication, centrifugation was performed at 4°C and 12000 rpm for 10 minutes, and the supernatant and precipitate were collected respectively. The supernatant and precipitate were added to 5× loading buffer, boiled in boiling water for 10 minutes, and identified by SDS-PAGE. The identification results are as follows: Figure 1 As shown in A, part of the recombinant p30 (rp30) protein is expressed in the supernatant, indicating that part of the rp30 protein is soluble. This part of the protein is purified by Ni-NTA affinity chromatography. After the protein is purified, SDS-PAGE is performed for identification. The identification results are shown in Figure 1 As shown in B.

[0070] Example 2 Preparation of ASFV p30 protein monoclonal antibody and amplification of antibody sequence

[0071] 2.1 Immunization of mice

[0072] Purified rp30 protein was used to immunize Babl / c mice at a dose of 30 μg / mouse. For the first immunization, the recombinant p30 protein was mixed with an equal volume of Freund's complete adjuvant and then emulsified, and multiple subcutaneous injections were performed. Reinforcement immunization was performed every 2 weeks, for a total of four immunizations. Recombinant p30 protein and Freund's incomplete adjuvant were mixed with an equal volume of emulsified, and the immunization method was the same as the first immunization.

[0073] 2.2 Preparation of monoclonal antibodies

[0074] Take the spleen cells of a mouse immunized with rp30 protein and mix them with SP20 cells (5:1 ratio), and then perform cell fusion under the action of fusion agent PEG. The fused cells are plated in a 96-well cell plate and cultured in a CO2 incubator at 37°C. When the hybridoma cells cover 1 / 10 of the bottom of the cell plate, take the supernatant for ELISA detection. The wells identified as positive by ELISA are then verified by indirect immunofluorescence test (IFA). Select the ELISA and IFA positive wells, and use the limiting dilution method to subclone the screened positive hybridoma cells. Observe under an inverted microscope, mark the wells where only a single clone grows, take the supernatant, and use the above ELISA and IFA methods for antibody detection. Positive cells enter the next round of subcloning, which is performed three times in total.

[0075] ELISA detection: The purified rp30 protein was diluted to 1 μg / mL with coating solution, and 50 μL / well was coated on the ELISA plate, coated at 37°C for 1 hour, washed four times with PBST (K2HPO4 0.26g, Na2HPO4·12H2O 2.89g, NaCl 8.50g, Tween-20 0.5mL, add water to 1L, pH7.4), and then blocked with 2% trehalose at 37°C for 1 hour. Take the hybridoma cell supernatant, 50 μL / well, act at 37°C for 30 minutes, wash four times with PBST, add HRP-labeled goat anti-mouse IgG (1:20000 dilution), act at 37°C for 30 minutes, wash four times with PBST, and use TMB for color development, and measure OD on the microplate reader. 450 .

[0076] IFA test: 1×10 4 MA104 cells were plated in a 96-well cell plate, and after the cells were fully plated, 0.1 MOI ASFV was inoculated. 48 hours after inoculation, the cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, and washed three times with 0.01 mol / L PBS (K2HPO4 0.26g, Na2HPO4·12H2O 2.89g, NaCl 8.50g, water was added to 1L, pH 7.4); then permeabilized with 0.1% TritonX-100, acted at room temperature for 10 minutes, and washed three times with PBS; then hybridoma cell supernatant and goat anti-mouse FITC-IgG were incubated, and the results were observed under a fluorescence microscope after the reaction was completed.

[0077] 2.3 Monoclonal Antibody Specificity Identification

[0078] After three subclonings, the cell supernatants secreted by the obtained single cell lines were subjected to IFA and western blot detection. The IFA identification results showed that the hybridoma cell 2A6 supernatant could detect specific green fluorescence signals ( Figure 2A), while no fluorescence signal was observed when the supernatant of SP2 / 0 cells was used to treat ASFV-infected cells ( Figure 2 B). Virus-infected and uninfected MA104 cells were collected, and SDS-PAGE was performed after cell lysis. The protein gel was then transferred to the NC membrane for western blot verification. The hybridoma cell 2A6 supernatant was used as the primary antibody and acted at room temperature for 1 hour. The anti-mouse HRP-IgG was used as the secondary antibody and acted at room temperature for 1 hour. After each action, it was washed 4 times with PBST and then exposed for color development. Western blot results showed that the hybridoma cell 2A6 supernatant could recognize the p30 protein in virus-infected cells ( Figure 3 ).

[0079] 2.4 Monoclonal Antibody Subclass Identification

[0080] According to Southern Biotech's SBA Clonotyping TM System / HRP Antibody Subclass Identification Kit Operating Instructions The antibody subclasses of the monoclonal antibodies produced by the hybridoma cell 2A6 were identified. Figure 4 As shown, the heavy chain constant region of 2A6 monoclonal antibody is of IgM type, and the light chain constant region is of Kappa type.

[0081] 2.5 Preparation and purification of ascites

[0082] Balb / c mice aged 10 to 12 weeks were selected and each mouse was intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. After 1 week, each mouse was intraperitoneally injected with 5×10 5 hybridoma cells (0.2 mL). After 7 to 10 days, the abdominal cavity of the mouse was obviously bulging. The ascites was collected and packaged. The ascites was purified by affinity chromatography according to the Protein L Spin Purification Kit of TAKARA Company. After purification, the purity was determined by SDS-PAGE electrophoresis ( Figure 5 ).

[0083] 2.7 PCR amplification and sequence determination of monoclonal antibody variable region genes

[0084] RNA of hybridoma cell 2A6 was extracted and reverse transcribed into cDNA using oligo-dt or random primers (PrimeScript II 1st Strand cDNA Synthesis Kit, TAKARA, 6210A). The amplification of antibody variable region genes was carried out according to the literature (von Boehmer, L., Liu, C., Ackerman, S., Gitlin, AD, Wang, Q., Gazumyan, A., Nussenzweig, MC, 2016. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nature protocols 11, 1908-1923.). After amplification, 1% agarose gel electrophoresis was performed. The heavy chain and kappa light chain variable region gene size was about 300 bp ( Figure 6 A and 6B), gel cutting to recover the target fragment. The recovered target fragment was inserted into the pMD-18T vector for sequencing. The sequencing results were compared and analyzed with the antibody gene library (IMGT). The sequencing results confirmed that the amplified sequence was the DNA of the heavy chain variable region and the light chain variable region of the monoclonal antibody.

[0085] Specifically, the DNA sequence encoding the heavy chain variable region of ASFV p30 protein monoclonal antibody 2A6 is shown in SEQ ID NO: 5; the DNA sequence encoding the light chain variable region of ASFV p30 protein monoclonal antibody 2A6 is shown in SEQ ID NO: 6. The amino acid sequence of the heavy chain variable region of ASFV p30 protein monoclonal antibody 2A6 is shown in SEQ ID NO: 3; the amino acid sequence of the light chain variable region of ASFV p30 protein monoclonal antibody 2A6 is shown in SEQ ID NO: 4. The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain and light chain variable regions of monoclonal antibody 2A6 are shown in Table 1 below.

[0086] Table 1 Amino acid sequences of heavy chain and light chain variable regions CDR1, CDR2 and CDR3 of monoclonal antibody 2A6

[0087] —— CDR1 CDR2 CDR3 Heavy chain VH GFSLTSYG IWSGGSTD AFYAMDY Light chain VL ESVDNYGISF AAS QQSKEVPWT

[0088] Example 3 Establishment of a competitive ELISA method for detecting ASFV p30 protein antibodies

[0089] 3.1 Selection of optimal reaction conditions for blocking ELISA

[0090] 3.1.1 Determination of the optimal antigen coating concentration and the optimal dilution of enzyme-labeled antibody

[0091] (1) ASFV p30 protein was diluted with coating solution at a ratio of 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL, and added horizontally to the ELISA plate, 100 μL was added to each well, and coated at 37°C for 1 hour. After drying, the plate was washed 4 times with PBST on a plate washer. The coating solution was 0.05 M carbonate buffer (1.5 g Na2CO3, 2.92 g NaHCO3 added to deionized water, pH adjusted to 9.6, and then fixed to 1 L).

[0092] (2) Add 2% trehalose as blocking solution and block at 37°C for 1 hour. After drying, wash 4 times using the same method as (1).

[0093] (3) Add the mixture of serum and enzyme-labeled antibody to the ELISA plate: a) Dilute the negative and positive sera with sample diluent at a ratio of 1:5; b) Dilute the enzyme-labeled antibody (HRP-2A6) with enzyme-labeled antibody diluent at a ratio of 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000; c) Mix the diluted serum and enzyme-labeled antibody at a ratio of 1:1 and add them vertically to the ELISA plate, 100 μL / well, at 37°C for 30 minutes. After drying, wash 4 times, using the same method as (1). The sample diluent was 1% BSA in 0.01M pH 7.4 PBS solution (0.2 g KH2PO4, 2.9 g Na2HPO4·12H2O, 8 g NaCl, 0.2 g KCl dissolved in 800 mL ultrapure water, adjusted to pH 7.4 with NaOH, and diluted to 1 L with water).

[0094] (4) Add 50 μL of TMB substrate colorimetric solution to each well and incubate at 37°C for 10 min.

[0095] (6) Add 50 μL 0.5 M H2SO4 to each well to terminate the reaction. Read the OD 450 nm value. Calculate the PI value, PI = (negative control OD value - serum OD value to be tested) / negative control OD value × 100%.

[0096] The results are shown in Table 2. When the PI value was the largest, the optimal coating concentration of ASFV p30 protein was 0.25 μg / mL, and the optimal dilution of the enzyme-labeled antibody was 4000 times.

[0097] Table 2 Selection of optimal coating concentration of antigen and optimal dilution of enzyme-labeled antibody

[0098]

[0099] 3.1.2 Determination of optimal coating conditions

[0100] The ELISA plate was coated with the optimal ASFV p30 protein antigen coating concentration determined in 3.1.1, and the coating was carried out at 37°C for 1h, 2h, 3h and 4h, and at 4°C overnight. 450 The results are shown in Table 3. When coated at 4°C overnight, the PI value of the detected sample is the largest, indicating that the optimal coating condition is 4°C overnight.

[0101] Table 3 Optimal coating conditions for antigens

[0102]

[0103] 3.1.3 Determination of the optimal blocking solution and reaction conditions

[0104] The ELISA plate was coated according to the optimal coating concentration and coating conditions of ASFV p30 protein determined in 3.1.1 and 3.1.2. 5% skim milk, 5% BSA or 2% trehalose was selected as the blocking solution and the plate was treated at 37°C for 1 hour. 450 The best blocking solution was determined by the nm value and PI value. The results are shown in Table 4. When blocked with 2% trehalose, the PI value of the tested sample was greater than that of 5% skim milk and 5% BSA, reaching 87.66%. Therefore, 2% trehalose has the best blocking effect. 5% skim milk, 5% BSA and 2% trehalose respectively represent the addition of 5g skim milk, 5g BSA and 2g trehalose to every 100mL PBST solution.

[0105] Table 4 Selection of the best blocking solution

[0106] Blocking solution 5% skim milk 5% BSA 2% Trehalose PI% 75.62 73.37 87.66

[0107] The ELISA plate was blocked with the optimal blocking solution, and blocking was performed at 37°C for 1 h, 2 h, 3 h, and 4 h, and at 4°C overnight. 450 The results are shown in Table 5. When the sample was blocked overnight at 4°C, the PI value of the sample was the largest, indicating that the best blocking condition was 4°C overnight.

[0108] Table 5 Determination of the optimal sealing time

[0109]

[0110] 3.1.4 Optimal dilution of serum to be tested

[0111] After coating and blocking the ELISA plate using the optimal conditions, the serum to be tested was diluted 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, and 1:512 times. As shown in Table 6, when the serum to be tested was diluted 1:4, the PI value was the largest, so the optimal dilution of the serum to be tested was 4-fold dilution.

[0112] Table 6 Determination of the action time of the serum to be tested

[0113] Serum dilution multiple 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 PI% 83.36 91.08 85.43 68.39 59.16 44.85 39.55 33.59 1.44

[0114] 3.1.5 Determination of reaction time

[0115] After the serum to be tested and the enzyme-labeled antibody were added to the ELISA plate, the plates were incubated at 37°C for 15 min, 30 min, 45 min, 60 min and 90 min. 450 The optimal reaction time was determined by the nm value and the PI value. As shown in Table 7, the PI value was the largest when the reaction was performed at 37°C for 30 min. Therefore, the optimal reaction condition was 37°C for 30 min.

[0116] Table 7 Optimal reaction time

[0117] Reaction time 15min 30min 45min 60min 90min PI% 89.31 94.58 93.31 93.02 90.92

[0118] 3.1.6 Determination of color development time

[0119] After the serum to be tested and the enzyme-labeled antibody are added to the ELISA plate, the reaction is carried out under the optimal reaction conditions, the ELISA plate is washed, and the color development is selected at 37°C for 5min, 10min, 15min and 20min. 450 The optimal substrate action time was determined by the nm value and the PI value. As shown in Table 8, when the color development time was 15 min, the PI value was the largest.

[0120] Table 8 Determination of color development time

[0121] Color development time 5min 10min 15min 20min PI% 92.91 94.31 95.14 95.02

[0122] 3.2 Determination of critical value

[0123] According to the optimized competitive ELISA method, 56 ASFV-positive serum samples and 128 ASFV-negative serum samples were tested, and their OD was measured after the test. 450 nm, and calculated the blocking rate (PI value), and used SPSS17.0 software to draw the ROC curve. When the Youden index was the largest (0.977), the specificity was 97.7%, the sensitivity was 100%, and the corresponding PI value was 46.30%. Therefore, when the PI value was ≥46.30%, it was judged as positive; when the sample PI value was <46.30%, it was judged as negative.

[0124] 3.3 Specificity test

[0125] The optimized competitive ELISA method was used to detect sera positive for classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), Seneca virus (SVA), pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), transmissible gastroenteritis virus (TGEV) and porcine parvovirus (PPV). Three replicates were set for each sample, and ASFV negative and positive sera were used as controls. Figure 7 As shown, using the optimized competitive ELISA method, only the ASFV serum sample was positive, and the others had no cross-reactions, indicating that the method established by the present invention has good specificity.

[0126] 3.4 Repeatability test

[0127] Eight serum samples (4 positive sera, P1, P2, P3 and P4; 4 negative sera, N1, N2, N3 and N4) were selected for testing to evaluate the intra- and inter-batch repeatability of the competitive ELISA method. 450 The value of nm is used to calculate the PI value. Then the coefficient of variation is calculated using the PI value. The results are shown in Table 9. The coefficients of variation of intra-batch and inter-batch repeatability tests were both below 10%, indicating that the established competitive ELISA method has good repeatability.

[0128] Table 9 Repeatability of competitive ELISA detection method

[0129]

[0130] 3.5 Detection results of the competitive ELISA method of the present invention on serum antibodies of experimental animals

[0131] The serum samples of 4 heads (No. 1, 2, 3 and 4) 1 to 11 days after the challenge and the serum samples of 4 heads (No. 5, 6, 7 and 8) not challenged 1 to 11 days were tested by the competitive ELISA method established by the present invention. The test results are shown in Figure 8 As shown, before infection, the serum antibodies of all the tested pigs were negative; on the 7th day after infection, the antibody of pig No. 6 was positive, on the 8th day after infection, the antibody of pigs No. 5 and 8 was positive, and on the 9th day after infection, the antibody of pig No. 7 was positive; this indicates that the competitive ELISA method established by the present invention can detect antibodies as early as the 7th day after virus infection.

[0132] 3.6 Comparative test between the competitive ELISA method of the present invention and the commercial kit

[0133] The ASFV p30 protein antibody competition ELISA method established by the present invention and a commercial kit (IDVet, France) were used to jointly detect 121 field pig serum samples, and then the compliance rate between the competitive ELISA method of the present invention and the test results of the commercial kit was calculated. The test results of the commercial kit showed that there were 42 positive sera and 79 negative sera; using the competitive ELISA method of the present invention, there were 44 positive sera and 77 negative sera. The total compliance rate was 96.69%, which was relatively high.

[0134] The two methods were further statistically analyzed and the Kappa value was calculated. The results showed that the Kappa value was 0.93 (Kappa ≥ 0.75), indicating that the two detection methods were almost completely consistent (Table 10). In summary, the ASFV p30 protein antibody competition ELISA method established by the present invention has a comparable detection effect to the commercial kit.

[0135] Table 10 Comparison results of competitive ELISA of the present invention and commercial kits

[0136]

[0137] The calculation formulas for the compliance rate and Kappa value are as follows:

[0138] Matching rate = [(a+d) / n]*100;

[0139] P A =(a+d) / n; P e =[(a+b)(a+c)+(c+d)(b+d)] / n 2 ; Kappa = (P A -P e ) / (1-P e ).

[0140] The embodiments described above are only preferred embodiments of the present invention and are not intended to limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made according to the structures, features and principles described in the patent scope of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. Monoclonal antibody against p30 protein of African swine fever virus, characterized in that: The heavy chain variable region of the monoclonal antibody includes CDR1 with an amino acid sequence of GFSLTSYG, CDR2 with an amino acid sequence of IWSGGSTD, and CDR3 with an amino acid sequence of AFYAMDY; the light chain variable region includes CDR1 with an amino acid sequence of ESVDNYGISF, CDR2 with an amino acid sequence of AAS, and CDR3 with an amino acid sequence of QQSKEVPWT.

2. The monoclonal antibody against p30 protein of African swine fever virus according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of the monoclonal antibody are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

3. The nucleotide encoding the monoclonal antibody against the African swine fever virus p30 protein according to claim 1, characterized in that: The nucleotide sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

4. A recombinant expression vector comprising the nucleotide sequence of claim 3.

5. A host cell comprising the recombinant expression vector according to claim 4.

6. Use of the monoclonal antibody according to claim 1 or 2, the nucleotide according to claim 3, the recombinant expression vector according to claim 4, and the host cell according to claim 5 in the preparation of an African swine fever virus detection reagent or kit.

7. A competitive ELISA kit for detecting antibodies against the P30 protein of African swine fever virus, characterized in that: include: An ELISA plate coated with African swine fever virus p30 protein, the monoclonal antibody according to claim 1 or 2 labeled with HRP, a positive control serum and a negative control serum.

8. The competitive ELISA kit according to claim 7, characterized in that It also includes coating solution, blocking solution, sample diluent, enzyme-labeled antibody diluent, washing solution, color developing solution and stop solution.

9. The competitive ELISA kit according to claim 8, characterized in that The coating solution is 0.05M carbonate buffer; The blocking solution was PBST solution containing 2% trehalose; The sample diluent was 1% BSA in 0.01 M PBS, pH 7.4; The enzyme-labeled antibody diluent was a 0.01 M pH 7.4 PBS solution containing 4% sucrose, 0.05% Tween-20, and 8% glycerol; The washing solution was 20-fold concentrated PBST; The color developing solution is TMB, and the stop solution is 0.5M H2SO4.

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