Generation of a sheep × mouse heterohybridoma cell line (1C6.3a6T.1D7) and evaluation of its use in the production of ovine monoclonal antibodies

Abstract

A stable aminopterin-sensitive sheep × mouse heterohybridoma cell line (1C6.3a6T.1D7) for use in the generation of sheep monoclonal antibodies is described. The line was first constructed by fusing the mouse myeloma line, NSO, to normal sheep lymphocytes obtained from the efferent lymphatic vessel of a cannulated popliteal lymph node. The line was rendered sensitive to aminopterin through a combination of irradiation and treatment with the anti-metabolite drug 6-thioguanine. Characterisation of the cloned cell line showed that it did not secrete sheep immunoglobulin (Ig) molecules, express Ig on the surface membrane, or express normal sheep B or T cell surface markers. The 1C6.3a6T.1D7 line has remained stable in tissue culture for over 2 years, showing no signs of reversion to aminopterin resistance. The 1C6.3a6T.1D7 cells have been used as fusion partners with lymphocytes from antigen primed sheep to generate sheep monoclonal antibodies to human chorionic gonadotropin (hCG) or a synthetic peptide analogue of the VP1 capsid protein of foot and mouth disease virus (FMDV). To optimise the efficiency of heterohybridoma generation, comparisons were made of peripheral blood, efferent lymph or excised lymph nodes as sources of antigen-stimulated lymphocytes for fusion. The results showed that lymphocytes prepared from either efferent lymph or lymph node on the fourth day following antigenic stimulation gave similar high fusion efficiencies, and both were vastly superior to peripheral blood lymphocytes. Results were also obtained which showed that the blast cells present in lymphoid tissues due to antigenic stimulation were the major cell types involved in the generation of viable antibody-secreting sheep × sheep × mouse heterohybridomas.