Preparation and identification of monoclonal antibodies against Cap protein of porcine circovirus type 2

The baculovirus-based Cap protein of porcine circovirus type 2(PCV2) was used to inoculate BALB/c mice.After 3 times of vaccinations,the splenocytes of the immunized mice were fused with murine myeloma cells SP2/0.After subcloning for 3 times,six positive hybridoma clones(McAbs) were screened by indirect enzyme-linked immunosorbent assay(ELISA),and were named as 3H9/1H5,3H9/3C7,4A9/3E2,4A9/3D7,6G7/7B6 and 6G7/8D3,respectively.Titers of the McAbs in supernatant of cell culture were from 1∶1 024 to 1∶2 048,and titers in ascites were from 1∶163 840 to 1∶204 800.The 3H9/1H5,3H9/3C7,6G7/7B6 and 6G7/8D3 belonged to IgG1,while 4A9/3E2 and 4A9/3D7 belonged to IgG2b.All of the McAbs had κ chain.Western-blotting analysis showed that all the McAbs could react with both of the recombinant Cap protein and the native PCV2 antigen.Results of indirect immunofluorescence assay indicated that they could react strongly with PCV2-infected PK15 cells.To map the epitopes recognized by the McAbs,five truncated fragments of PCV2 Cap proteins(51-150aa,51-200aa,51-234aa,101-200aa,101-234aa)expressed in Escherichia coli were used for Western-blotting analysis,and the results showed that 4A9/3E2 and 4A9/3D7 could recognize the epitope within 101-150aa,while the other four McAbs might recognize the epitope within 1-50aa or conformational epitope.McAbs constructed in the present study provided new diagnostic methods for the studies of pathogenesis of PCV2 in future.