Production of monoclonal antibodies specific to vitellogenin and zona radiata proteins in greenback mullet, Liza subviridis

Abstract

Monoclonal antibodies (MAbs) specific to vitellogenin (VTG) and zona radiata protein (ZRP) of Greenback Mullet (Liza subviridis) were generated using proteins extracted from unfertilized eggs as immunogens. Hybridomas producing antibodies that bound to VTG and ZRP in both native and denatured forms were selected by dot-blotting and were characterized for their specificities by Western blot analysis and Immunohistochemical staining. Eleven MAbs specific to VTG can be grouped into 6 categories according to their binding capabilities to antigens. Most of MAbs bound to both native and denatured antigens while one group of them (VTG-313, 530) bound to only native antigens and 2 groups of monoclonal antibodies were able to cross react to vitellogenic cells by immunohistochemical staining. Isotype and subisotype of antibodies were identified by sandwich ELISA. All MAbs belong to the IgG₁ isotype except one that belongs to the IgG₂ isotype. For the production of monoclonal antibodies specific to ZRP, 11 antibodies were specific to both native and denatured forms of ZRP in blood serum of mature female mullets. ZRP can be grouped into 4 categories according to their binding capabilities to different stages of antigens and all monoclonal antibodies belong to the IgG₁ isotype. Indirect immunoperoxidase competitive ELISA using VTG and ZRP monoclonal antibodies specific to VTG and ZRP levels in juvenile mullets injected with various estradiol (E2) were determined. The result indicated that the levels of VTG and ZRP were significantly increased in responding to the increase level of E2. This indicates that detection of VTG and ZRP in Greenback Mullet using antibodies is an effective method for determining xenoestrogenic effect in water.