Monoclonal antibodies against sea bassDicentrarchus labrax(L.) immunoglobulins: immunolocalisation of immunoglobulin-bearing cells and applicability in immunoassays

Sea bass immunoglobulins were single-step purified from the whole serum by affinity chromatography on protein A-sepharose. The purified immuno-globulins had an apparent molecular weight of 78 kDa (heavy chain) and 27 kDa (light chain) in SDS-PAGE. Immunoglobulins were used as immunogens in mice, and after fusion, the hybridomas were screened by indirect immunofluorescence on living cells, western blotting, and immunohistochemical staining of Bouin fixed tissues. Among the positive hybridomas obtained, three clones were selected according to their ability to recognise either the immunoglobulin light chain (DLIg3) or the heavy chain (DLIg13 and DLIg14). DLIg3 stained in FACS analysis 1% of cells in the whole blood and 19·7% of PBL, 1·1% in the thymus, 22·2% in the 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 FACS, whereas recognised fixed cells following ABC-immunoperoxidase staining of spleen, head-kidney and midgut. DLIg3 was used to set up a sensitive ELISA assay to detect and quantify purified and serum immunoglobulins. Using purified immunoglobulins the assay sensitivity resulted 1·2 ng ml−1, whereas the serum immunoglobulin content resulted 3·92±2·16 mg ml−1with an assay detection limit of 3·9 ng ml−1, corresponding to a serum dilution of 9·7×105times.