Immunopurification of T-cells from sea bass Dicentrarchus labrax (L.)

The monoclonal antibody DLT15, specific for thymocytes and peripheral T-cel ls of the teleost fish Dicentrarchus labrax: (sea bass), was used to purify immunoreactive cells from blood and gut-associated lymphoid tissue. The pu rification was performed by immune-magnetic sorting of leucocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by cytofluorimetric analysis. Following a sing le step, the percentage of DLT15-purified cells was 88 +/- 10% for gut-asso ciated lymphoid tissue and 79 +/- 18% for blood leucocytes. DLT15-purified cells from gut-associated lymphoid tissue were employed for RNA extraction and cDNA synthesis. In RT-PCR experiments using as primers degenerate oligo nucleotides corresponding to the peptide sequence MYWY and VYFCA of the tro ut TcR beta chain, a 203 bp product was amplified. When sequenced, the cDNA was found to show 60% nucleotide identity to the tr out TcRV beta 3. By 3' -RACE the cDNA was elongated to obtain the TcR constant region, with high s imilarity to other fish TcR sequences. These results strongly suggest that cells recognised by DLT15 are putative T lymphocytes. (C) 2000 Academic Pre ss.