Development of Monoclonal Antibodies against Classical Swine Fever Virus E2 Protein by Single B Cell Sorting Technology and Its Application in ELISA
The purpose of this study was to develop a classical swine fever virus(CSFV)E2 mon-oclonal antibody-based competition ELISA(Enzyme Linked Immunosorbent Assay)for evalua-ting the efficiency of the live attenuated C-strain and E2 subunit vaccines.Firstly,the E2 gene of CSFV was constructed into pFast BAC vector,and the E2 protein was efficiently expressed in SF9 cells;Secondly,6-8 weeks of BABL/c mice were immunized at intervals with purified E2 protein,and the single B cells were then sorted out at the gate of IgM-/E2+by flow cytometry.Then,the heavy and light chains of E2 antigen-specific IgG antibodies were amplified by semi-nested PCR,after sequencing,the heavy and light chains of the antibodies were constructed into pCDNA3.1vector,and then were co-transfected into HEK293 cells to prepare the monoclonal an-tibodies against CSFV E2.The results showed that the two monoclonal antibodies derived from single B cells,namely mAb3A9(IgG1,kappa)and mAb4F7(IgG2a,lambda),could efficiently reacted with the linear epitopes25 GLTTTWKEYSHDLQL39 and259 GNTTVKVHASDERGP273 of CSFV E2 protein,respectively.In addition,the competitive ELISAs developed using the mono-clonal antibodies and E2 protein mentioned above exhibited an excellent diagnostic sensitivity(97.49％,95.97％)and specificity(96.08％,94.38％)in the process of detecting serum sam-ples,which provides a favorable technical support for the gradual elimination of CSF in China.
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