Development And Application Of A Double Antibody Sandwich ELISA For Detection Of African Swine Fever

African swine fever（ASF）is an infectious disease caused by African swine fever virus（ASFV）,which is highly contagious,hemorrhagic and high mortality in pigs.Given that there is currently no safe and effective vaccine,sensitive and rapid diagnosis of it is the key to effectively prevent and control the disease.African swine fever virus is a huge and complex DNA virus.The main shell protein P72 is one of the most important structures of the virus particle.P72 protein has a high degree of immunogenicity and reaction,and is one of the key antigen in ASF diagnosis.In this study,antibodies of the main structural protein P72 of African swine fever virus are planned to establish a dual antibody sandwich antigen detection method.The research results are reported as follows:1.Expression and purification of recombinant p72 and p54 proteins of ASFVThe specific primers were designed to amplify the p72 protein and p54 protein genes by PCR,and the gene fragments of 1900 bp and 555 bp were obtained respectively.The amplified products were cloned into the prokaryotic expression plasmid p ET-32a by in-fusion seamless connection technology to construct recombinant plasmids p ET32a-p72 and p ET32a-p54.The two recombinant plasmids were transformed into competent E.coli expression strain BL21（DE3）and induced by 0.1 m M/L and 1.0 m M/LIPTG,respectively.Highly purified recombinant p72and p54 proteins were obtained by Ni column purification.2.Preparation of monoclonal antibody and HRP labelingBALB/c mice were immunized with purified recombinant p72 protein and p54 protein,and spleen cells were fused with SP2/0 tumor cells.Three monoclonal antibodies against p72 protein（4HF8,H6H5,6D3）and three monoclonal antibodies against p54 protein（9C3,6H6,9G9）were screened by indirect ELISA.They were labeled with horseradish peroxidase（HRP）,and the reactogenicity and specificity of the three monoclonal antibodies were verified by Indirect immunofluorescence,Western Blot and direct ELISA.3.Preliminary establishment and application of double antibody sandwich ELISA antigen detection method for African swine feverThe ASFV double antibody sandwich ELISA method was established by using ASF positive pig serum Ig G as capture antibody and horseradish peroxidase（HRP）p72 monoclonal antibody as detection antibody,and the reaction conditions were optimized.Through experiments,the optimal conditions for the sandwich ELISA method were determined:the capture antibody coating concentration was 2.5μg/ml,the detection antibody was diluted 1:1500 times,the best blocking agent was 5%BSA,and the color development time was15 min.The sensitivity,specificity and repeatability of the established ASFV double antibody sandwich ELISA method were tested.The recombinant p72 protein was used as the standard to fit the antigen standard curve.The effective linear range was 0.1μg/ml-25μg/ml,and the sensitivity was 0.1μg/ml.The standard curve of virus titer was fitted with the inactivated African swine fever virus with known titer as the standard,and the sensitivity was 10^3.7 TCID₅₀.There was no cross-reaction with CSFV,PRV,PRRSV,PPV and PCV2.Repeatability test showed that the coefficient of variation of OD_450nm value was less than 5%.The detection of different clinical samples proved that the method has good specificity,repeatability and sensitivity.The antigens in spleen,submandibular lymph nodes,whole blood and serum can be detected in clinical tissue samples,and the sensitivity of whole blood test results is the highest.It has good consistency with q PCR method.Eight virus samples with known virus titers were tested,and the TCID₅₀error rate was less than 10%.This method was used to quantitatively detect the content of inactivated antigen of ASFV inactivated by different inactivation methods and mixed with different adjuvants.The results showed that the sensitivity of the sample could be improved afterβ-propiolactone inactivation,and different adjuvants would have different effects on the detection.The detection of different clinical samples proved that the method has good specificity,repeatability and sensitivity.Eight virus samples with known virus titers were tested,and the TCID₅₀ error rate was less than 10%.The antigens in spleen,submandibular lymph nodes,whole blood and serum can be detected in clinical tissue samples,and the sensitivity of whole blood test results is the highest.In summary,this study used recombinant p72 protein-specific monoclonal antibody and ASFV positive serum Ig G to establish a double antibody sandwich ELISA for African swine fever virus antigen detection method with good sensitivity,specificity and repeatability.This method provides an effective method for the diagnosis of ASFV infection and the quantitative detection and efficacy evaluation of ASFV inactivated vaccine in the future.