A short-duration polyethylene glycol fusion technique for increasing production of monoclonal antibody-secreting hybridomas

Abstract

In this study the exposure period of the lymphocyte-myeloma cell mixture to the fusogen was evaluated for its influence upon the yield of total hybridoma colonies and those which secreted monoclonal antibodies. Sp2/0 and FOX-NY myeloma cells were fused for varying periods with murine splenic lymphocytes immunized with sheep red blood cells. The optimal fusion period consisted of adding the fusogen (5.0 ml Kodak 1450 PEG, 0.5 ml dimethylsulfoxide, and 4.5 ml of phosphate-buffered saline, pH 7.0) to the cell mixture over a 45 s period at 37°C. The fusion process was stopped by gradually diluting the mixture in 50 ml of RPMI-1640. After 10 min, the cells were centrifuged, resuspended in selective medium with feeder macrophages and cultured. In comparison to common, longer fusion techniques, this procedure produces approxiamately a 5-fold increase in the number of hybrids produced when using the Sp2/0 cells and a 30-fold increase in the number of hybrids produced when using the FOX-NY cells as the fusion partner. In both cases, virtually all the wells contain monoclonal antibody-secreting hybridoma colonies. This high efficiency fusion technique can be used most advantageously to produce monoclonal antibodies against weak immunogens or to reduce the time needed for immunonization with stronger immunogens.