| Seneca virus A(Senecavirus A,SVA)is a new type of Picornavirus,which is classified into the genus Seneca virus by the International Committee on Virus Classification and named as "Seneca virus A".The virus has four structural proteins,which are VP1,VP2,VP3 and VP4.Studies have shown that VP1,VP2 and VP3 proteins can all induce strong immune responses,among which VP2 protein has the highest immunogenicity.In addition,from the molecular level,the structural protein VP2 has the largest number of amino acids and contains the main neutralization epitopes of SVA.Studies have confirmed that VP2 is more conservative than VP1.The indirect ELISA detection methods constructed with VP1,VP2 and VP3 proteins also showed that VP2 protein had higher affinity,and VP2 protein was the preferred target protein for serological detection of SVA.In this study,monoclonal antibodies specific to SVA VP2 were prepared by immunizing BALB/c mice with prokaryotic expression of SVA VP2 protein,and the detection method of SVA blocking ELISA antibody was successfully established.The specific research contents are as follows:1.Preparation and identification of Monoclonal Antibodies against Seneca virus A VP2 proteinThe soluble expression conditions of SVA VP2 were optimized by E.coli prokaryotic expression system,and the soluble expression of VP2 recombinant protein was successfully carried out.Western blot analysis and mouse immunoassay showed that the recombinant protein had good specificity and immunogenicity.Five 6-week-old BALB/c female mice were immunized with the recombinant protein,and five hybridoma cell lines stably secreting m Abs against SVA VP2 protein were successfully obtained,which were named1G7,3E3,4E4,6B2 and 6H8,respectively.After continuous culture for 20 generations,the titer of antibody was basically stable,the titer of ELISA antibody in the supernatant of the cell culture were in the range of 1:1600～1:6400,and the titer of antibody of the ascites were in the range of 1:128000～1:4096000.The identification of antibody subtypes showed that all the heavy chains of the monoclonal antibodies were Ig G1,and the light chains were Kappa chains.Western blot and IFA results showed that the Mc Abs secreted by 5hybridoma cell lines could react specifically with SVA virus and recombinant VP2 protein.These Mc Abs will lay an important foundation for the identification of their subsequent epitopes and the development of antibody differential diagnosis kit.2.Establishment of a blocking ELISA detection method for Seneca virus A with monoclonal antibodies VP2 proteinA blocking ELISA method for detecting SVA VP2 antibody was established by using purified recombinant VP2 protein as a coating antigen and horseradish peroxidase(HRP)labeled anti-VP2 protein monomer.The optimized reaction conditions were as follows:Antigen coating concentration was 0.25μg/m L,incubation at 37°C for 2 hours and then at4°C overnight;the best blocking condition was 1 hour incubation at 37°C with 1% BSA;the best action condition for the serum detection was sample dilution at 1:1 and 1.5 hours incubation at 37°C;the best working condition for MAb-HRP(1:1000 dilution)was 45 minutes incubation at 37°C;The best action time of TMB is 10～15 minutes at 37°C.The diagnostic criteria of this method was as follows: positive when the percentage inhibition(PI)≥ 41.25%,negative when the PI≤225.29%,and inconclusive when 25.29%< PI <41.25%.The method has no cross-reactions with positive sera of common swine diseases,and the variation coefficients were both less than 11%.The sensitivity test showed that the positive detection rate of this method was 98.3%,and the minimum detection titer of positive serum was 1:8.The coincidence rate with neutralization test is 70.1%.The results of 506 clinical pig serum samples showed that the positive rate of SVA antibody was 74.5%.In conclusion,the blocking ELISA method developed in this study has good specificity and stability,and can be used to detect SVA antibody in serum. |
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