Development and characterization of a monoclonal antibody against the putative T cells of Labeo rohita.

Abstract

In this study, we have described the development and characterization of monoclonal antibodies (MAbs) directed against thymocytes of rohu, Labeo rohita. MAbs were obtained by immunizing BALB/c mice with freshly isolated and nylon wool column enriched mononuclear cells of thymus. Positive clones against thymocytes were screened by cellular ELISA. The hybridoma showing strong reactivity with nylon wool enriched mononuclear cells, and non-reactivity with a rohu thymus macrophage cell line and rohu serum was selected and subjected to single cell cloning by limiting dilution. The MAbs secreted by a positive clone were designated as E6 MAb. Western blotting of reduced protein from enriched thymocytes showed that E6 reacted with a 166.2kDa polypeptide and belongs to the IgG1 subclass. Flow cytometric analysis of gated lymphocytes, revealed that the percentage of E6 positive (E6+) cells in thymus (n=5, 720.4±79.70g) was 89.7%. Similarly, the percentage of E6+ cells in kidney, spleen and blood (n=5) was 6.71, 1.71 and 1.88%, respectively. In indirect immunoperoxidase test, E6+ cells appeared to be lymphoid cells with a high nucleus to cytoplasmic ratio and were densely packed in the central region of thymus whereas, a few cells were found to be positive in kidney and spleen sections. E6 MAb also reacted with a small population of lymphocytes in blood smear. This MAb appears to be a suitable marker for T lymphocytes and can be a valuable tool in studying immune response and ontogeny of L. rohita immune system.