Development, characterization of monoclonal antibodies specific for the ORF25 membrane protein of Cyprinid herpesvirus 2 and their applications in immunodiagnosis and neutralization of virus infection

Cyprinid herpesvirus 2 (CyHV-2) is the causative agent of haematopoietic necrosis disease in farmed gibel carp (Carassius auratus gibelio). It appears to be spreading cross the world, and has resulted in massive mortality and great economic losses. Although many sensitive nucleic acid-based diagnostic methods have been developed, effective immunodiagnosis and neutralization approaches based on monoclonal antibodies (MAbs) against CyHV-2 membrane protein are still scarce to study. In this study, ORF25 gene was chemically synthesized and cloned into a pET-28a expression vector to produce rORF25 (recombinant ORF25) in E. coli BL21 (DE3). The purified rORF25 was served as an immunogen to prepare monoclonal antibodies (MAbs), and five efficient hybridoma cell lines were selected by enzyme-linked immunosorbent assay (ELISA). Among them, MAbs 7H10-1B6 and 1F8-1B6 belonged to the IgG2b isotype, while the other three MAbs designed as 8D9-1H6, 3H2-1G5 and 2C3-1E6 belonged to the IgG1 isotype. Western blotting results indicated that MAbs could specifically identify the rORF25. MAb 2C3-1E6 could specifically detect ORF25 in CyHV-2 infected Fathead Minnow cells and gible carp tissues by ELISA, indirect immunofluorescence and immunohistochemistry. In vitro neutralization assay showed that MAb 2C3-1E6 exhibited a titer-dependent neutralization effect on CyHV-2 infection in FHM cells. The results indicated that MAbs against rORF25 could serve as effective detection probes and potential neutralizing antibody for haematopoietic necrosis disease in gibel carp.