Using HTS-ELISA method to make anti SP-10 couple monoclonal antibody

Objective To prepare specificity recognize different epitopes of SP-10 couple monoclonal antibodies.Methods Balb/C mice were immunized with SP-10, and screened secret anti-SP-10 monoclonal antibody hybridoma by High Throughput Screening ELISA（HTS-ELISA）and prepared monoclonal antibodies. Then HRP labeling was used to analyze the characteristics of monoclonal antibodies recognizing different epitopes of SP-10. Results Three hybridoma cell lines were screened with monoclonal antibodies secreting specific recognition of SP-10 protein. The monoclonal antibodies secreted by them were named mAb80, mAb81 and mAb208, in which the titer of mAb80 and mAb81 was more than 1:1×107 and the titer of mAb208 was 1:2×106, and the epitopes of mAb208 recognizing SP-10 were significantly different from those of mAb80 and mAb81, showing that they were couple monoclonal antibodies. A sandwich ELISA for the detection of SP-10 was constructed by using mAb208 labeled HRP as the detection antibody and mAb81 as the capture antibody. The minimum detection limit of SP-10 was up to 0.01 ng/mL, the linear range was 0.01-10 ng/mL, and the ELISA recovery of SP-10 with 0.05, 0.5, 5 ng/mL was between 99.1% and 106.7%. Conclusion The anti-SP-10 paired monoclonal antibody prepared by this method can provide the antibody material basis for the establishment of spermatozoa protein SP-10 immune detection system.