Cloning and expression of scpb protein of Streptococcus agalactiae from Tilapia and establishment of indirect ELISA for IgM antibody detection

In order to establish an ELISA method with good specificity, sensitivity, repeatability and accuracy for detecting Streptococcus agalactis from tilapia, firstly, the ScpB gene of Streptococcus agalactiae from Tilapia was cloned, expressed and purified. Then, the indirect ELISA method was established by using the recombinant protein as the coating antigen, with the Tilapia serum for test as the primary antibody, the anti-tilapia IgM monoclonal antibody 2C10 as the secondary antibody, and the goat anti-mouse IgG-HRP as the tertiary antibody. The conditions of the method（antigen coating mass concentration, primary antibody dilution, antigen coating conditions, blocking solution, primary antibody action time, secondary antibody action time） were optimized. Finally, the critical value of the method was determined and the specificity, sensitivity, repeatability and compliance tests were carried out.The results showed that the size of the purified ScpB protein of Streptococcus agalactiae from Tilapia was 104.6 ku, which was consistent with the expected size and could be recognized by Tilapia IgM specific antibody. The optimal antigen-coated mass concentration of the established IgM antibody indirect ELISA method was 0.1 μg/mL; the optimal primary antibody dilution was 1∶5; the optimal antigen-coated condition was 4 ℃ for 12 h; the optimal sealing solution was 5% skim milk powder; the optimal primary antibody action time was 2 h and the optimal secondary antibody action time was 0.5 h. The upper limit of critical value was 0.456, and the lower limit of critical value was 0.377. The intra-assay coefficient of variation was 3.349%-5.272%, and the inter-assay coefficient of variation was 2.793%-5.902%, both of which were lower than 10.000%. The positive serum of Streptococcus agalactiae from Tilapia and the immune serum of Tilapia ScpB protein could only be detected, but the positive serum of Aeromonas hydrophila from Tilapia, the positive serum of Pseudomonas fluorescens from Tilapia, the positive serum of Streptococcus iniae from Tilapia and the positive serum of Vibrio anguillarum from Tilapia could not be detected. The maximum dilution of the tested serum was 1∶640. The positive rate was slightly lower than that of the whole Streptococcus agalactiae coated by ELISA,and the coincidence rate was 83.43%. The results indicated that the Streptococcus agalactia IgM antibody from Tilapia indirect ELISA method had good specificity, sensitivity, repeatability and accuracy.