Immunopurification of B lymphocytes from sea bass Dicentrarchus labrax (L.).
The monoclonal antibody DLIg3, specific for immunoglobulins and B cells of the teleost fish Dicentrarchus labrax (Mediterranean sea bass), was used to enrich immunoreactive cells from peripheral blood, spleen, and head kidney leukocytes. The purification was performed by immunomagnetic sorting of leukocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by immunofluorescence and cytofluorimetric analysis. Following a single immunopurification step, the percentages of DLlg3-purified cells were 61% +/- 6% from peripheral blood leukocytes, 66% +/- 5% from splenocytes, and 77% +/- 9% from head kidney cells. DLIg3-immunopurified cells, from the head kidney of antigen-primed fish displayed an enhanced proliferation index when incubated with the immunization antigen. DLIg3-purified cells from head kidney lymphoid tissue were employed for RNA extraction and complementary DNA synthesis, and in reverse transcriptase polymerase chain reaction experiments using specific primers corresponding to the sequences of the sea bass immunoglobulin light chain, and of T-cell receptor. DLIg3-purified cells displayed enhanced expression of the immunoglobulin gene, and lower expression of T-cell receptor.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology*; D001492:Bass / Q000235:genetics / Q000276:immunology*; D003720:Densitometry; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D007147:Immunoglobulin Light Chains / Q000276:immunology; D018189:Immunomagnetic Separation; D066298:In Vitro Techniques; D007668:Kidney / Q000276:immunology; D007962:Leukocytes / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013154:Spleen / Q000276:immunology
D000911:Antibodies, Monoclonal; D007147:Immunoglobulin Light Chains; D011948:Receptors, Antigen, T-Cell