MONOCLONAL-ANTIBODIES AGAINST SEA BASS DICENTRARCHUS-LABRAX (L) IMMUNOGLOBULINS - IMMUNOLOCALIZATION OF IMMUNOGLOBULIN-BEARING CELLS AND APPLICABILITY IN IMMUNOASSAYS

Sea bass immunoglobulins were single-step purified from the whole seru m by affinity chromatography on protein A-sepharose. The purified immu noglobulins had an apparent molecular weight of 78 kDa (heavy chain) a nd 27 kDa (light chain) in SDS-PAGE. Immunoglobulins were used as immu nogens in mice, and after fusion, the hybridomas were screened by indi rect immunofluorescence on living cells, western blotting, and immunoh istochemical staining of Bouin fixed tissues. Among the positive hybri domas obtained, three clones were selected according to their ability to recognise either the immunoglobulin light chain (DLIg3) or the heav y chain (DLIg13 and DLIg14). DLIg3 stained in FAGS analysis 1% of cell s in the whole blood and 19.7% of PBL, 1.1% in the thymus, 22.2% in th e spleen, 12.2% in the head-kidney, and 1.5% in the midgut. DLIg13 and DLIg14 were unable to stain living cells by immunofluorescence and FA GS, whereas recognised fixed cells following ABC-immunoperoxidase stai ning of spleen, head-kidney and midgut. DLIg3 was used to set up a sen sitive ELISA assay to detect and quantify purified and serum immunoglo bulins. Using purified immunoglobulins the assay sensitivity resulted 1.2 ng ml(-1), whereas the serum immunoglobulin content resulted 3.92 +/- 2.16 mg ml(-1) with an assay detection limit of 3.9 ng ml(-1), cor responding to a serum dilution of 9.7 x 10(5) times. (C) 1996 Academic Press Limited.