Development Of Indirect ELISA And Blocking ELISA For Detection Of African Swine Fever Virus Antibody

African Swine Fever(ASF)is a highly infectious animal disease caused by the African Swine Fever Virus(ASFV).Its main clinical symptoms are superficial skin congestion,cyanosis,and visceral bleeding of pigs.ASFV belongs to the ASFV family and has 24 genotypes.It is a linear double-stranded DNA virus with tetrahedral structure,with a total length of 170-190 kb,including 151 reading frames and an envelope,encoding nearly 200 proteins in total.As one of the structural proteins of ASFV,p54protein is located on the inner side of the virus particle with a molecular weight of about29 k Da.P54 is a good immunogen with strong conserved properties and can be used as a detection antigen for ASFV antibody.The outbreak of African Swine Fever has brought huge economic losses to China.In addition,ASFV infection can be determined by positive antibody detection when there is no vaccine at present.Therefore,this study focuses on ASFV antibody detection technology,selects p54 as the detection antigen,and establishes an indirect ELISA method to detect ASFV antibody.Since the sheep anti-pig IgG-HRP used by indirect ELISA method can be widely used for detection of antibodies against other swine pathogens,it is easy to produce cross-reaction or false positive detection results.Subsequently,this study used ASFV p54 protein to prepare anti-p54monoclonal antibody,and established a blocking ELISA method.It is committed to providing the most suitable test method for the clinical detection of ASF,as well as technical support for the diagnosis and prevention of ASF.The main research contents are as follows:1.Prokaryotic expression of ASFV p54 protein and establishment of indirect ELISA methodThe recombinant plasmid pET32a(+)-p54 was obtained by effectively predicting and truncating the ASFV p54 protein and embedding it into the prokaryotic vector pET32a(+).The recombinant plasmid was then transformed into Escherichia coli BL21.Monoclony was selected and induced by IPTG,ultrasonic crushing and affinity chromatography to obtain ASFV p54-HIS.The purification effect and immunogenicity of the protein were verified by SDS-PAGE and western blot.Finally,the coated antigen p54-HIS for the ELISA method was obtained.To acquire the indirect ELISA method,this study is to establish a complete square matrix is used to determine the p54-HIS best dilution ratio,sheep against swine IgG-HRP dilution multiple and the best serum dilution ratio,then optimize the ELISA method in each step of the reaction time and temperature,the end results are:on the dilution ratio:p54-HIS is 1:5000(0.3?g/m L),Enzyme-labeled secondary antibody is 1:5000,virus serum is 1:40;The reaction time and temperature were 30 min and 37?,respectively.The determination criteria were as follows:S/P?0.25 was positive for ASFV antibody;S/P<0.25 was negative for ASFV antibody.Then,the reproducibility,specificity,and coincidence rate of the method were verified.The results showed that the method established in this study was highly reproducible.Compared with a commercial kit,indirect ELISA method is similar in specificity.And the coincidence rate is as high as 100%,which has clinical value.2.Preparation of monoclonal antibody against p54 protein and establishment of blocking ELISA methodThis study the expression of p54-HIS protein has good immunogenicity,preparation of monoclonal antibodies as immunogen to immune BALB/c mice,in the process protein p54-GST is expressed to screen positive clones,which is expressed by prokaryotic expression system with the recombinant plasmid pET42b(+)-p54.Through cell fusion,double label antigen screening positive clones,three times of cloning,ascites preparation,and purification of ascites,finally we acquire the 3 strains resistant p54monoclonal antibody,then through ELISA detection of 3 strains of monoclonal antibody titer,finally choose one of the highest titer of single fight 4-4C,and then the 4-4C-HRP sheet resistance as a follow-up study.To a series of 4-4C single resistance feature validation:4-4C types identified as IgG₁ and lg?;Its titer was 1.6×10⁶;ELISA,western blot and IFA showed that 4-4C could specifically recognize ASFV.A blocking ELISA method was developed to detect ASFV antibody using p54-HIS protein and monoclonal antibody 4-4C-HRP.The results showed that the dilution ratio of p54-HIS was 1:4000(0.38?g/m L),the dilution ratio of monoclonal antibody 4-4C-HRP was 1:22000(0.15?g/m L),the optimal dilution ratio of serum to be tested was 1:1,the reaction time of serum and antibody was 30 min,and the temperature was 37?.The criteria for ASFV antibody positivity are the blocking rate of the sample to be tested?50%;otherwise,ASFV antibody negative is determined.Then,the sensitivity,specificity and coincidence rate of the blocking ELISA method and the commercial kit were compared in parallel.The results showed that the sensitivity of the blocking ELISA method in this study was1:64,while the sensitivity of the commercial kit was 1:32.In terms of specificity and coincidence rate,the results of this study are comparable to the commercial kit,providing technical support for the detection of ASF.