Characterisation of monoclonal antibodies specific for sea bass (Dicentrarchus labraxL.) IgM indicates the existence of B cell subpopulations

Three monoclonal antibodies (MAb) against sea bass IgM (Dicentrarchus labraxL.) were selected (WDI 1–3) based on ELISA, Western blot and FACS analysis. Further characterisation was done by immunofluorescence on cytocentrifuge slides and immuno-electron microscopy. All MAb were found to be of the IgG1/|gkisotype and were effective in detectingVibrio anguillarum-ordalii antibody titres with an ELISA. Under denaturing conditions in Western blots, all MAb reacted with a tetrameric, dimeric and monomeric form of serum IgM. Under reducing conditions WDI 1 recognises the heavy (H) chain (±78 kDa) and both WDI 2 (slightly) and WDI 3 (strongly) recognise the light (L) chain (±28 kDa) in Western blots of whole serum and Protein A isolated sea bass IgM. The average percentages (N=6) of surface IgM+(B) cells in peripheral blood leucocytes (PBL), head kidney, trunk kidney, spleen, thymus and gut for the different MAb were estimated by FACS. The highest was in PBL (33%) stained with WDI 3 and the lowest in thymus (1%). All MAb showed specific immunogold labelling of a subpopulation of lymphoid cells in both PBL and spleen and of plasma cells and some macrophages in spleen. Additional percentage staining in FACS analysis using different combinations of the three MAb, as well as double immunofluorescence staining of cytocentrifuge preparations and Coomassie blue staining of SDS–PAGE of Protein A purified IgM under reduced conditions, strongly suggest the existence of at least two H and L chains expressed in different combinations in at least two subpopulations of B cells in sea bass. From these results it is also concluded that the combination of WDI 1 with WDI 3 stains all or almost all B cells.