Development of monoclonal antibodies against serum immunoglobulins from gibel carp (Carassius auratus gibelio) and their applications in serodiagnosis of inapparent infection and evaluation of vaccination strategies

The disease outbreak caused by viral infection and bacterial pathogens has hampered the sustainable development of the gibel carp (Carassius auratus gibelio) industry, lack of monoclonal antibodies against serum immunoglobulin (Ig) of gibel carp has impeded the development of nonfatal immunoassays in detection of pathogen infection and understanding of fish immune response post vaccination. In the present study, serum Ig of gibel carp was purified by a combination of salting-out and DEAE Sepharose Column chromatography. The purified Ig had an apparent molecular weight of 74 kDa and 24 kDa in SDS-PAGE. Three monoclonal antibodies (MAbs) against Ig, designed as 2F4-1G10, 3H3-1E8 and 7H11-1C8, were developed with purified Ig preparations, which were selected on the basis of the indirect enzyme-linked immunosorbent assay (ELISA). Western blotting showed that MAbs 2F4-1G10 and 7H11-1C8 reacted with the heavy chain of IgM, while MAb 3H3-1E8 showed a reaction with the light chain. MAb 7H11-1C8 could react with surface Ig-positive (sIg +) lymphocytes under indirect immunofluorescence assay. Fluorescence-activated cell sorter analysis revealed that the percentage of slg + lymphocytes were 32.68% in peripheral blood and 12.13% in pronephros. MAb 7H11-1C8 was proven to be effective in detecting the Cyprinid Herpesvirus 2-specific serum Ig, and determining the levels of Aeromonas hydrophila specific Ig in serum and immune organs under different vaccination strategies.