CN115947795B - Recombinant protein for detecting ASFV antibody, double-antigen sandwich ELISA kit and application thereof - Google Patents
Recombinant protein for detecting ASFV antibody, double-antigen sandwich ELISA kit and application thereof Download PDFInfo
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Abstract
The invention relates to the field of biotechnology, in particular to a recombinant protein for detecting an African Swine Fever Virus (ASFV) antibody, a double-antigen sandwich ELISA kit and application thereof, wherein the kit comprises an ELISA plate coated with antigen, an ELISA antibody, african swine fever positive control serum, an ASF negative control serum, a diluent, a reaction solution, a 10X washing solution, a TMB substrate solution and a stop solution. The antigen of the coated ELISA plate in the kit is recombinant ASFV p54 and p30 fusion protein (p 54-30-His) carrying His tag. The kit can be used for serodiagnosis of ASFV infection, monitoring of antibody level, epidemiological investigation and the like, can effectively avoid interference of protein in a detection mark of a recombinant subunit vaccine or virus-like particle vaccine immune pig antibody, and provides an accurate and effective method for ASFV antibody detection and detection.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a recombinant protein for detecting ASFV antibodies, a double-antigen sandwich ELISA kit and application thereof.
Background
African swine fever (African swine fever, ASF) is a highly contagious, febrile disease caused by African swine fever virus (African swine fever virus, ASFV) in pigs or wild boars, and highly pathogenic strains can cause 100% death in infected pigs.
Currently existing diagnostic techniques for ASF mainly include three major categories: nucleic acid detection, antigen detection, and antibody detection. The virulent strain often causes acute death of pigs, and the detection difficulty of antibodies; after infection of medium and low virulence strains, the long disease course can stimulate the organism to generate better humoral immune response, the detection of the antibody is one of effective diagnosis methods, OIE also clearly indicates that the recovered pig or the pig with continuous infection for a period of time has ASFV antibody, and whether the pig is infected with ASFV can be confirmed by the detection of the antibody.
The ASFV antibody detection method mainly comprises ELISA, including indirect ELISA, blocking ELISA/competition ELISA, etc. The indirect ELISA is often a recombinant antigen, the detection sensitivity is higher, the preparation of the kit is relatively simple, fusion tag proteins (such as His tags) are used in the purification of the recombinant antigen, while virus-like particle (VLPs) subunit vaccines used in clinical pigs are also often used for purifying the antigen in the preparation of the subunit vaccines, a certain level of tag protein antibodies are generated in normal pigs after multiple immunizations, and the detection results are easy to be false positive despite the fact that serum is greatly diluted in the detection process; the blocking ELISA/competition ELISA needs to use enzyme-labeled specific antibody, ASFV antibody in pig serum is combined with antigen on an ELISA plate, the adsorption of the enzyme-labeled antibody is blocked, and the negative and positive of a detection result are determined through the chromogenic of a substrate solution.
Disclosure of Invention
In order to achieve the aim of the invention, the recombinant protein p54-30-His is used for coating an ELISA plate, anti-ASFV p54 and p30 antibodies of a sample to be detected are adsorbed through antigen-antibody reaction, reaction liquid is added, the indirectly adsorbed antibodies and recombinant protein GST-p54-30 are combined with GST antigen adsorbed on the ELISA plate by using HRP-labeled anti-GST ELISA antibody, after the substrate solution develops color, the substrate solution is compared with a critical value, whether the ASFV antibodies exist in the sample to be detected is judged, and a double antigen sandwich ELISA kit for detecting the ASFV antibodies is established and assembled based on the principle. The kit can be used for serodiagnosis of ASFV infection, monitoring of antibody level, epidemiological investigation and the like, can effectively avoid interference of protein in detection of the recombinant subunit vaccine or virus-like particle (VLPs) vaccine immune pig antibody, and provides an accurate and effective method for ASFV antibody detection and detection.
The invention adopts the technical scheme that:
a recombinant protein for detecting ASFV antibodies takes ASFV p54 and p30 as antigens, the sequence of the recombinant protein is shown as SEQ ID No.1, and the amino acid sequence of the ASFV antigen is shown as SEQ ID No. 2.
The invention also provides a preparation method of the recombinant protein for detecting the ASFV antibody, which comprises the steps of respectively introducing restriction enzyme cutting sites NdeI and XhoI at two ends of p54 and p30 genes, and inserting the recombinant protein into a vector pUC57 after synthesis.
A double-antigen sandwich ELISA kit for detecting ASFV antibody is characterized by comprising recombinant protein for detecting ASFV antibody, an ELISA plate, an HRP-labeled anti-GST enzyme antibody, ASF positive control serum, ASF negative control serum, a diluent, a reaction solution, 10X washing solution, TMB substrate solution and a stop solution.
Furthermore, the antigen of the 96-hole ELISA plate coated in the kit is recombinant ASFV p54 and p30 recombinant protein p54-30-His carrying His tag; the reaction solution is the recombinant ASFV p54 and p30 recombinant protein GST-p54-30 with GST tag in working concentration.
Furthermore, the recombinant protein p54-30-His is obtained by expressing and purifying plasmid pET-30a (+) loaded with the recombinant protein in BL21 (DE 3) escherichia coli; the recombinant protein GST-P54-30 is obtained by expressing and purifying plasmid pGEX-6P-1 loaded with the recombinant protein in BL21 (DE 3) escherichia coli.
Further, the HRP-labeled anti-GST enzyme-labeled antibody is an HRP-labeled mouse-derived anti-GST monoclonal antibody; the ASF positive control serum is serum prepared by immunizing pigs with recombinant p54 and p30 fusion proteins; the ASF negative control serum is healthy pig serum.
Further, the diluent is 5% calf serum containing 0.01% proclin300;
the 10 x washing solution is: 80g of NaCl, 2g of KCl and Na 2 HPO 4 14.4g，KH 2 PO 4 2.4g of Tween-20 0.5mL,Proclin300 0.1mL, dissolving in 800mL of distilled water, adjusting pH to 7.4, fixing the volume to 1L, filtering with a 0.22 μm filter, sterilizing, and preserving at 4 ℃;
the termination liquid is 2M H 2 SO 4 . The double-antigen sandwich ELISA kit for detecting ASFV antibody comprises a 96-hole ELISA plate coated with recombinant ASFV p54 and p30 fusion proteins carrying His labels, a reaction solution of recombinant ASFV p54 and p30 fusion proteins carrying GST labels, and an HRP-labeled anti-GST monoclonal antibodyEnzyme-labeled antibody, ASFV positive control serum prepared by immunizing pigs with recombinant p54 and p30 fusion proteins and ASFV negative control serum from healthy pigs.
The invention also provides a preparation method of the double-antigen sandwich ELISA kit for detecting ASFV antibodies, which is characterized by comprising the following steps:
(1) Recombinant protein p54-30-His and GST-p54-30 are prepared;
(2) Preparing ASF positive control serum and ASF negative control serum;
(3) Preparation of double-antigen sandwich ELISA kit for detecting ASFV antibody.
Further, the amplification primer sequences adopted for detecting the recombinant protein of the ASFV antibody are shown as SEQ ID NO.3 and SEQ ID NO. 4.
The invention also provides application of the recombinant protein and the kit in preparation of a reagent for detecting the African swine fever virus antibody.
The preparation method of the double-antigen sandwich ELISA kit for detecting ASFV antibody comprises the following steps:
(1) Recombinant protein production
artificial synthesis of asfv p54, p30 genes: optimizing ASFV p54 and p30 gene codons to make them suitable for expression in colibacillus, respectively introducing restriction enzyme cutting sites NdeI and XhoI at two ends of p54 and p30 gene to synthesize the gene, its sequence is shown in SEQ ID NO.1, and the synthesized gene is cloned into vector pUC57;
b. recombinant bacterium construction: the plasmid pET30a (+) and ASFV p54 and p30 genes in pUC57 are respectively digested with restriction enzymes NdeI and XhoI, the digested plasmid pET30a (+) and about 726bp ASFV p54 and p30 genes are recovered, the recovered fragments are connected by T4 DNA ligase, the connection products are transformed into BL21 (DE 3) escherichia coli competent cells, and recombinant bacteria carrying the plasmid pET-5430 are screened out by adopting a NdeI and XhoI double digestion method; plasmid pET-5430 is used as template, and primer is used
‘F 5’-CGCggatccCTATTCTCTTCAAGAAAG-3’(SEQ ID NO.3)，
R5'-GCAActcgagTTTTTTTTTCAGCAGTTTG-3' (SEQ ID NO. 4) PCR-amplified ASFV P54 and P30 genes, restriction enzyme BamHI and XhoI are used to cleave PCR products and pGEX-6P-1 plasmid respectively, the digested pGEX-6P-1 plasmid and about 726bp ASFV P54 and P30 genes are recovered by a nucleic acid recovery kit, T4 DNA ligase is used to connect the recovered fragments, the connection products are transformed into BL21 (DE 3) E.coli competent cells, and recombinant bacteria carrying plasmid pGEX-5430 are cleaved and screened by adopting a BamHI and XhoI double enzyme method.
ASFV p54, p30 recombinant fusion protein expression, purification: recombinant bacteria carrying plasmids pET-5430 and pGEX-5430 were inoculated with a 2 XYT medium containing 50. Mu.g/mL kanamycin and 100. Mu.g/mL ampicillin at a volume ratio of 1:100, and cultured at 37℃to OD 600 =0.8, 1mmol/L IPTG was added and induced for 4h at 37 ℃; centrifuging at 8000rpm for 2min, washing the obtained thallus with 0.01M PBS solution for 2 times, and centrifuging again; splitting the thalli obtained by re-centrifugation by using an ultrasonic instrument, wherein the power of the ultrasonic instrument is 50W,10 s/time, and the total time is 10min; centrifuging at 12000rpm for 20min at 4deg.C, and collecting precipitate; purifying recombinant proteins p54-30-His and GST-p54-30 by using Ni-NTA Agarose and GST Binding Resin respectively; and (3) taking the purified recombinant fusion protein, carrying out 2 mug/channel, carrying out 12% polyacrylamide gel electrophoresis, transferring the recombinant fusion protein to a nitrocellulose membrane by a protein transfer printer, and carrying out Western-blotting verification by using monoclonal antibodies of anti-ASFV p30 and anti-ASFV p 54.
(2) Control serum preparation:
asf positive control serum: regulating the concentration of the recombinant protein p54-30-His to 1mg/mL by using PBS (phosphate buffer solution) with pH of 7.4 and 0.01M, mixing with Freund's complete adjuvant with equal volume, fully emulsifying by using a cytolytic instrument, and intramuscular injecting the emulsified recombinant protein into healthy pigs with the age of 45 days and 2 mL/pig; on day 10 after the first immunization, boost 1 with the same dose of antigen mixed and emulsified with Freund's incomplete adjuvant; boosting 1 time after 10 days with the same dose of recombinant protein; aseptically collecting pig blood and separating serum on day 7 after the third immunization; according to the conventional indirect ELISA method, 2.5 mug/mL recombinant protein p54-30-His is respectively used for coating an ELISA plate, the ELISA plate is blocked and then reacts with pig separation serum which is diluted 10 times continuously, a secondary antibody is an HRP-marked goat anti-pig IgG (H+L) antibody which is diluted to the working concentration, a substrate is TMB substrate solution, and a stop solution is 2M H 2 SO 4 Detection of absorbance per well at 450nm wavelengthELISA antibody titer of 1:10 of recombinant fusion protein immune pig serum is determined 6 Serum was stored at-20 ℃ after sub-packaging and used as ASF positive control serum for detection.
Asf negative control serum: collecting blood of 75-day-old healthy pigs, separating serum, subpackaging, and preserving at-20deg.C to obtain ASF negative control serum for detection.
(3) Preparation of double-antigen sandwich ELISA kit for ASFV antibody:
a. ELISA plate 2: diluting recombinant protein p54-30-His with pH9.6 and 0.05M carbonate buffer solution to a final concentration of 1 mug/mL, adding 100 mug of the recombinant protein p54-30-His into each hole of a 96-well plate, standing at 37 ℃ for 2h, washing with a washing solution PBST of pH7.4 for 2 times, adding 200 mug of a sealing solution into each hole, standing at 37 ℃ for 1h, washing with the washing solution PBST for 2 times, beating residual water on absorbent paper, air-drying in an ultra-clean workbench, filling into an aluminum foil bag, vacuumizing and sealing, and preserving at 4 ℃. Wherein the PBST component of the washing solution is 0.01M PBS,0.5 per mill Tween-20 and 0.01 percent Proclin300; the blocking solution was PBST, pH7.6, which was a washing solution containing 5% calf serum.
b. Dilution liquid: 30mL of 5% calf serum containing 0.01% Proclin300, and the specific preparation method is as follows: 5mL calf serum and 10 mu L Proclin300 are completely dissolved in PBST, the final volume is fixed to 100mL, a 0.22 mu m filter is used for filtration and sterilization, 30mL is taken out and packaged into a sterilization bottle, and the sterilization bottle is preserved at 4 ℃.
c. The reaction solution: recombinant protein GST-p54-30 was diluted to 1. Mu.g/mL with the diluent, filtered and sterilized with a 0.22 μm filter, and placed in a 25mL sterilization flask for storage at 4 ℃.
d. Enzyme-labeled antibody: HRP-labeled mouse-derived anti-GST monoclonal antibody 10 μl, stored at-20 ℃;
ASF positive control serum, ASF negative control serum: 1mL each, stored at-20 ℃.
f.10×washing: PBST 30mL which is 10 times concentrated, the preparation method is as follows: 80g of NaCl, 2g of KCl and Na 2 HPO 4 14.4g，KH 2 PO 4 2.4g of Tween-20 0.5mL,Proclin300 0.1mL, dissolved in 800mL of distilled water, pH7.4 adjusted, constant volume to 1L, filtration and sterilization by a 0.22 mu m filter, and filling 30mL into a sterilizing bottle for preservation at 4 ℃.
TMB substrate solution: TMB substrate solution 25mL, stored at 4 ℃.
h. Stop solution: 2M H 2 SO 4 15mL, stored at room temperature.
Advantageous effects
During the early experiments, we found that His-tagged recombinant antigen had His-tag interference phenomenon (His antibodies were present in pigs immunized with multiple virus-like particle vaccines) when the sow serum was tested. Recently, we find that p30 can form dimer in the experimental process, so that the p30 amino terminal sequence in the invention only uses carboxyl terminal part with high antigen index, removes dimer formation region, effectively avoids false positive in the double antigen sandwich ELISA antibody detection process, and the p54 antigen used removes hydrophobic signal peptide region, thereby effectively improving recombinant protein expression quantity; expressing and purifying ASFV p54 and p30 fusion recombinant proteins with excellent antigenicity and respectively carrying His tag and GST tag by a genetic engineering method, coating an ELISA plate by the His tag recombinant proteins, adding serum to be detected, binding ASFV antibody in the serum to the ELISA plate, adding ASFV recombinant protein carrying GST tag, capturing the protein by the antibody bound on the ELISA plate, binding the HRP-labeled anti-GST monoclonal antibody with the indirect-bound GST tag, and determining the existence of the ASFV antibody after developing by a substrate solution. The method not only can detect IgG antibodies, but also can detect IgM antibodies produced by organisms in early stage, and most importantly eliminates the interference of common fusion tag proteins, and serum to be detected does not need to be diluted in a large amount, and has low background, thus having great significance for accurate detection of ASFV antibodies.
Drawings
FIG. 1 is a diagram showing immunoblotting of recombinant ASFV p54, p30 fusion proteins carrying different tags; wherein M is Prestained Protein Marker; 1. 2 are respectively the immunoblotting results of purified recombinant proteins GST-p54-30, p54-30-His and anti-ASFV p30 monoclonal antibodies; 3. 4 are respectively the purified recombinant proteins GST-p54-30, p54-30-His and the serum immunoblotting result of healthy mice; 5. 6 are the immunoblotting results of purified recombinant proteins GST-p54-30, p54-30-His and anti-ASFV p54 monoclonal antibody, respectively.
Detailed Description
For the purpose of illustrating the technical scheme and technical purposes of the present invention, the present invention is further described below with reference to the accompanying drawings and detailed description.
Biological material source:
pET30a (+): coli expression vector, purchased from Novogen corporation (Cat No. 69909.3) in the united states;
BL21 (DE 3) E.coli (E.coli) competent cells: purchased from sea-based biology (HaiGene) technology Co., ltd (Cat No. K10127);
ASFV antibody positive serum: 12 ASFV antibody positive sera were provided by the national african swine fever reference laboratory at the center of china animal health and epidemiology;
ASFV antibody negative serum: 444 ASFV antibody negative serum in 2017, porcine Parvovirus (PPV) positive serum, porcine foot-and-mouth disease virus (FMDV) positive serum, porcine Reproductive and Respiratory Syndrome Virus (PRRSV) positive serum, porcine circovirus type 2 (PCV 2) positive serum and porcine pseudorabies virus (PRV) positive serum are identified and stored by the laboratory.
Enzyme-labeled antibody: an HRP-labeled mouse-derived anti-GST monoclonal antibody was purchased from ATAGENT XLABORATORIES (Cat No. ATMA10044 Mo), an HRP-labeled goat anti-pig IgG (H+L) antibody was purchased from Abcam (Cat No. ab6915), and an HRP-labeled goat anti-mouse IgG (H+L) antibody was purchased from Proteintech (Cat No. S00001).
Example 1 recombinant protein preparation
Artificial synthesis of asfv p54, p30 genes: the codons of the ASFV p54 and p30 genes are optimized to be suitable for expression in escherichia coli, restriction enzyme sites NdeI and XhoI are respectively introduced at two ends of the p54 and p30 genes to synthesize the genes, and the genes are inserted into a vector pUC57, and the gene sequences are as follows:
catatgCTATTCTCTTCAAGAAAGAAAAAAGCTGCTGCCGCTATTGAGGAGGAAGATATACAGTTTATAAATCCTTATCAAGATCAGCAATGGGCAGAAGTCACTCCACAACCAGGTACCTCTAAACCGGCTGGAGCGACTACAGCAAGTGCAGGCAAACCAGTCACGGGCAGACCGGCAACAAACAGACCAGCAACAAACAAACCAGTCACGGACAACCCAGTTACGGACAGACTAGTCATGGCAACTGGCGGGCCAGCGGCCGCACCTGCGGCCGCGAGTGCTCATCCGACTGAGCCTTACACGACAGTCACTACTCAGAACACTGCTTCACAAACAATGTCGGCTATTGAAAATTTACGACAAAGAAACACCTATACGCATAAAGACCTAGAAAACTCCTTGGATATCGAAGAAGAAACCGAATCTTCTGCTTCTTCTGAATCTATCCACGAAAAAAACGACAACGAAACCAACGAATGCACCTCTTCTTTCGAAACCCTGTTCGAACAGGAACCGTCTTCTGAAGAACCGAAAGACTCTAAACTGTACATGCTGGCTCAGAAAACCGTTCAGCACATCGAACAGTACGGTAAAGCTCCGGACTTCAACAAAGTTATCCGTGCTCACAACTTCATCCAGACCATCCACGGTACCCCGCTGAAAGAAGAAGAAAAAGAAGTTGTTCGTCTGATGGTTATCAAACTGCTGAAAAAAAAActcgag (SEQ ID NO. 1) encodes an ASFV antigen having the amino acid sequence LFSSRKKKAAAAIEEEDIQFINPYQDQQWAEVTPQPGTSKPAGATTASAGKPVTGRPATNRPATNKPVTDNPVTDRLVMATGGPAAAPAAASAHPTEPYTTVTTQNTASQTMSAIENLRQRNTYTHKDLENSLDIEEETESSASSESIHEKNDNETNECTSSFETLFEQEPSSEEPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIHGTPLKEEEKEVVRLMVIKLLKKK (SEQ ID NO. 2), wherein aa1-aa133 are the p54 proteins with the signal peptide removed and aa134-aa 238 are the p30 carboxy-terminal proteins with the dimer forming region removed.
2. Recombinant bacterium construction: the ASFV p54 and p30 genes in pET30a (+) plasmid and pUC57 are digested with restriction enzymes NdeI and XhoI respectively, the digested pET30a (+) plasmid and about 726bp ASFV p54 and p30 genes are recovered, the recovered fragments are connected by T4 DNA ligase, the connection products are transformed into BL21 (DE 3) escherichia coli competent cells, and the recombinant plasmid is selected by NdeI and XhoI double digestion and named pET-5430; plasmid pET-5430 is used as template, and primer is used
F 5’-CGCggatccCTATTCTCTTCAAGAAAG-3(SEQ ID NO.3)，
The ASFV P54 and P30 genes were amplified by PCR with R5'-GCAActcgagTTTTTTTTTCAGCAGTTTG-3' (SEQ ID NO. 4), digested with restriction enzymes BamHI and XhoI, pGEX-6P-1 plasmid and about 726bp ASFV P54 and P30 genes were recovered, the recovered fragments were ligated with T4 DNA ligase, the ligation products transformed into BL21 (DE 3) E.coli competent cells, and recombinant plasmids were selected by double digestion with BamHI and XhoI, and named pGEX-5430.
ASFV p54, p30 recombinant fusion protein expression, purification: recombinant bacteria carrying plasmids pET-5430 and pGEX-5430 were inoculated with a 2 XYT medium containing 50. Mu.g/mL kanamycin and 100. Mu.g/mL ampicillin at a volume ratio of 1:100, and cultured at 37℃to OD 600 =0.8, 1mmol/L IPTG was added and induced for 4h at 37 ℃; centrifuging at 8000rpm for 2min,washing the thalli obtained by centrifugation with 0.01M PBS solution for 2 times, and centrifuging again; splitting the thalli obtained by re-centrifugation by using an ultrasonic instrument, wherein the power of the ultrasonic instrument is 50W,10 s/time, and the total time is 10min; centrifuging at 12000rpm for 20min at 4deg.C, and collecting precipitate; purifying the recombinant protein with His tag (p 54-30-His) and the recombinant protein with GST tag (GST-p 54-30) by using Ni-NTA Agarose and GST Binding Resin respectively; the purified recombinant fusion protein was separated by electrophoresis on a 12% polyacrylamide gel at 2. Mu.g/channel, transferred to nitrocellulose membrane by a protein transfer instrument, and subjected to Western-blotting assay using monoclonal antibodies against ASFV p30 and ASFV p54 (FIG. 1).
EXAMPLE 2 preparation of Positive control serum and negative control
Asf positive control serum: the concentration of the purified p54-30-His recombinant protein is adjusted to 1mg/mL by using PBS with pH of 7.4 and 0.01M, and the mixture is mixed with Freund's complete adjuvant with equal volume and fully emulsified in a tissue refiner; intramuscular injection of the recombinant fusion protein of water-in-oil into healthy pigs of 45 days old, 2 mL/pig; 10 days after the first immunization, boost 1 with the same dose of antigen mixed with Freund's incomplete adjuvant; boosting 1 day after 10 days with the same dose of recombinant fusion protein; aseptically collecting pig blood and separating serum on day 7 after the third immunization; according to the conventional indirect ELISA method, 2.5 mug/mL recombinant protein p54-30-His is respectively used for coating an ELISA plate, the ELISA plate is blocked and then reacts with pig separation serum which is diluted 10 times continuously, a secondary antibody is an HRP-marked goat anti-pig IgG antibody which is diluted to the working concentration, a substrate is TMB substrate solution, and a stop solution is 2M H 2 SO 4 Detecting the absorbance value of each hole at the wavelength of 450nm, and determining the ELISA antibody titer of the recombinant fusion protein immune pig serum to be 1:10 6 Serum was stored at-20 ℃ after sub-packaging and used as ASF positive control serum for detection.
Asf negative control serum: collecting blood of 75-day-old healthy pigs, separating serum, subpackaging, and preserving at-20deg.C to obtain ASF negative control serum for detection.
EXAMPLE 3 establishment of double antigen sandwich ELISA method of ASFV antibody
Optimization of ELISA reaction conditions: coating an ELISA plate with recombinant protein p54-30-His at different concentrations, sealing with different types of sealing solutions (comprising 5% skim milk, 2% BSA, 5% calf serum, 5% gelatin and the like), carrying out combined reaction on p54-30 antiserum with different dilution factors and negative control serum, and carrying out optimization on reaction time at the same time, wherein the final determination is that: the effect is best when the recombinant protein p54-30-His is diluted by 0.05M carbonate buffer solution (pH 9.6) to 8 mug/mL, the ELISA plate is coated for 2h at 37 ℃,5% calf serum is sealed for 45min at 37 ℃, the serum is diluted 5 times for 45min at 37 ℃,1 mug/mL GST-p54-30 is reacted for 45min, and TMB substrate solution is reacted for 15 min.
ELISA threshold and determination of experiment establishment conditions: 200 ASFV-negative sera were tested according to the above-described optimized reaction conditions, and absorbance (OD 450 ) And calculate the average value of the readingsAnd standard deviation SD, in->The threshold value of the ELISA method was finally determined to be 0.091.ASFV positive control serum and ASFV negative control serum were repeated 10 times with the above method, respectively, and the average value +.>And standard deviation of>Value sum->The value is taken as a condition critical value for establishing positive and negative control serum, and the result shows that the positive control serum OD 450 The requirement is more than or equal to 1.510, and the negative control serum OD 450 Less than or equal to 0.087.
Example 4 preparation of double antigen sandwich ELISA kit for ASFV antibody
1. Antibody detection elisa plate: the kit contains 2 treated 96-well ELISA plates (aluminum foil bags), and the ELISA plate treatment process is as follows: the recombinant protein p54-30-His was diluted with 0.05M carbonate buffer (pH 9.6) to a final concentration of 1. Mu.g/mL, 100. Mu.L was added to each well of the 96-well plate, incubated at 37℃for 2h, washed 2 times with wash solution PBST (0.01 MPBS, 0.5%Proclin 300, pH 7.4), blocked at 37℃for 1h by adding blocking solution (0.01M PBS,5% calf serum, 0.01% Proclin300, pH 7.6), washed 3 times with wash solution PBST, blotted with absorbent paper to remove residual moisture, air-dried in an ultra clean bench, and the ELISA plate and the desiccant were placed in an aluminum foil bag (1 ELISA plate/bag), vacuum-sealed, and stored at 4 ℃.
2. Dilution liquid: 30mL of 5% calf serum containing 0.01% Proclin300, and the specific preparation method is as follows: completely dissolving 5mL calf serum and 10 mu LProclin300 in PBST, fixing the final volume to 100mL, filtering with 0.22 mu m filter, sterilizing, taking 30mL, filling into a sterilizing bottle, and preserving at 4 ℃;
3. the reaction solution: diluting recombinant protein GST-p54-30 to 1 mug/mL with a diluent, filtering and sterilizing with a 0.22 mu m filter, filling into a 25mL sterilizing bottle, and preserving at 4 ℃;
4. enzyme-labeled antibody: HRP-labeled mouse-derived anti-GST monoclonal antibody 10 μl, stored at-20 ℃;
asfv positive control serum, ASF negative control serum: 1mL each, stored at-20deg.C;
6.10 Xwashing: PBST 30mL which is 10 times concentrated, the preparation method is as follows: 80g of NaCl, 2g of KCl and Na 2 HPO 4 14.4g，KH 2 PO 4 2.4g of Tween-20 0.5mL,Proclin300 0.1mL, dissolving in 800mL of distilled water, regulating pH to 7.4, fixing the volume to 1L, filtering with a 0.22 μm filter, sterilizing, taking 30mL of the solution, filling into a sterilizing bottle, and preserving at 4 ℃;
tmb substrate solution: 25mL of TMB substrate solution, and preserving at 4 ℃;
8. stop solution: 2M H 2 SO 4 15mL, stored at room temperature.
And bottling and assembling the aseptic components to obtain the double-antigen sandwich ELISA kit.
EXAMPLE 5 use of double antigen sandwich ELISA kit of ASFV antibody
1. And taking out the ELISA plate from the kit, balancing to room temperature (16-26 ℃) and avoiding direct sunlight.
2. Diluting serum to be detected and ASFV antibody positive and negative control serum with a dilution ratio of 5 times, respectively adding samples into corresponding holes of an ELISA plate, and allowing the samples to act at 37 ℃ for 45min at 100 mu L/hole;
washing the ELISA plate with a washing solution of 200 mu L/well x 3 times for 3 min/time, and drying the liquid in the well on absorbent paper;
4. adding 100 mu L of reaction solution per hole, and reacting at 37 ℃ for 45min;
5. adding enzyme-labeled antibody diluted by 1:10000, 100 mu L/hole, and reacting at 37 ℃ for 45min;
washing the ELISA plate with a washing solution of 200 mu L/well x 3 times for 3 min/time, and drying the liquid in the well on absorbent paper;
7. adding TMB substrate solution, 100 mu L/hole, and standing at room temperature in dark place for 15min;
8. adding a stopping solution to stop the reaction, wherein the concentration of the stopping solution is 50 mu L/hole;
9. the enzyme label instrument reads the wavelength absorbance value (OD 450) of 450nm of each hole;
10. in accordance with ASFV positive serum control well OD 450 1.510, ASFV positive serum control well OD 450 Under the precondition of less than or equal to 0.087, the OD of the serum sample to be detected 450 > 0.091, judged ASFV antibody positive; OD (optical density) 450 And less than or equal to 0.091, and determining that the ASFV antibody is negative.
Example 6 double antigen Sandwich ELISA kit specificity test for ASFV antibody
The prepared double antigen sandwich ELISA kit for detecting ASFV antibody is used for detecting the positive serum of common porcine viral diseases, including Porcine Parvovirus (PPV) positive serum, porcine Foot and Mouth Disease Virus (FMDV) positive serum, porcine Reproductive and Respiratory Syndrome Virus (PRRSV) positive serum, porcine circovirus type 2 (PCV 2) positive serum and porcine pseudorabies virus (PRV) positive serum, and determining whether the kit has cross reaction with the serum or not, and the result shows that the sample detection is negative (Table 1).
Table 1 results of specific assay of kit
Example 7 clinical application of double antigen Sandwich ELISA kit of ASFV antibody
456 clinical serum samples were tested using the ASFV antibody ELISA kit of the invention, of which 12 ASFV antibody-positive serum and 444 ASFV antibody-negative serum. The results show that the detection results of 12 ASFV antibody positive serum are positive, and the accuracy of the antibody positive detection is 100%; of 444 ASFV antibody negative serum, 425 parts of detection results are negative, and the antibody negative detection accuracy is 95.7%; the overall detection compliance was 95.8%.
Example 8 in-batch and batch-to-batch reproducibility assay of double antigen sandwich ELISA kit for ASFV antibodies
1. Batch repeatability test: randomly extracting 3 ELISA plates in the same batch, balancing at room temperature for 30min, detecting 20 parts of ASFV antibody-negative pig serum and 9 parts of ASFV antibody-positive serum according to the established double-antigen sandwich ELISA method, and detecting according to OD 450 The values calculated the Coefficient of Variation (CV) within the batch, which showed CV.ltoreq.7.18% (Table 2).
TABLE 2 results of in-batch repeated tests of ASFV antibody ELISA test kit
2. Batch-to-batch repeatability test: 1 ELISA plate of different batches is randomly extracted, the plates are balanced for 30min at room temperature, 20 parts of ASFV antibody-negative pig serum and 9 parts of ASFV antibody-positive serum are detected according to an established double-antigen sandwich ELISA method, and the detection is carried out according to OD 450 The values calculated the coefficient of variation between batches, which showed CV.ltoreq.6.56% (Table 3).
TABLE 3 results of repeated tests between batches of ASFV antibody ELISA test kit
The foregoing has shown and described the basic principles and main features of the present invention and the advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (6)
1. The double-antigen sandwich ELISA kit for detecting ASFV antibody is characterized by comprising an ELISA plate, an HRP-labeled anti-GST enzyme-labeled antibody, ASF positive control serum, ASF negative control serum, a diluent, a reaction solution, 10X washing solution, TMB substrate solution and stop solution; the antigen of the coated ELISA plate in the kit is ASFV p54 and p30 fusion protein carrying His tag, and is named as recombinant protein p54-30-His; the reaction solution comprises ASFV p54 and p30 fusion proteins carrying GST tags, which are named as recombinant proteins GST-p54-30; the gene sequence of the fusion protein of ASFV p54 and p30 is shown in SEQ ID No. 1.
2. The double antigen sandwich ELISA kit for detecting ASFV antibody according to claim 1, wherein the recombinant protein p54-30-His is obtained by expressing and purifying plasmid pET-30a (+) loaded with the recombinant protein in BL21 (DE 3) escherichia coli; the recombinant protein GST-P54-30 is obtained by expressing and purifying plasmid pGEX-6P-1 loaded with the recombinant protein in BL21 (DE 3) escherichia coli.
3. The double-antigen sandwich ELISA kit for detecting ASFV antibody according to claim 1, wherein the HRP-labeled anti-GST enzyme-labeled antibody is an HRP-labeled mouse-derived anti-GST monoclonal antibody; the ASF positive control serum is serum prepared by immunizing pigs with recombinant p54 and p30 fusion proteins; the ASF negative control serum is healthy pig serum.
4. A double antigen sandwich ELISA kit for detecting ASFV antibodies according to claim 1,
the diluent is 5% calf serum containing 0.01% procrin300;
the 10 x washing solution is: 80g of NaCl, 2g of KCl and Na 2 HPO 4 14.4g，KH 2 PO 4 2.4g of Tween-20 0.5mL,Proclin300 0.1mL, dissolving in 800mL of distilled water, adjusting pH to 7.4, fixing the volume to 1L, filtering and sterilizing by a 0.22 mu m filter, and preserving at 4 ℃;
the termination liquid is 2M H 2 SO 4 。
5. The method for preparing the double-antigen sandwich ELISA kit for detecting ASFV antibodies according to any one of claims 1-4, which is characterized by comprising the following steps:
recombinant protein p54-30-His and GST-p54-30 are prepared;
preparing ASF positive control serum and ASF negative control serum;
preparation of double-antigen sandwich ELISA kit for detecting ASFV antibody.
6. The use of the kit of claim 1 for preparing a reagent for detecting african swine fever virus antibodies.
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