Affiliations: [a] Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA | [b] Institute of Biotechnology, University of Wurzburg, Wurzburg, Germany
Note: [] Address reprint requests to: Steven K.H. Foung, M.D., Stanford University Blood Bank, 800 Welch Road, Palo Alto, CA 94304, USA.
Abstract: Hypoosmolar conditions have permitted the development of electrofusion techniques capable of producing human hybridomas from as few as 105 B cells. A hybridoma formation efficiency of one hybrid for each 125 input B cells has been achieved with Epstein-Barr virus-activated B cells and mouse–human heteromyelomas. This is at least 100-fold higher in efficiency than with polyethylene glycol-induced cell fusion, as well as a 50- to 100-fold decrease in the required number of human B cells. The ability to fuse a small number of input B cells should lead to a greater success rate in immortalizing the rare antigen-specific B cells. The critical parameters include fusion voltages, the composition and number of wash steps used in cell preparation, the composition and duration of exposure to hypoosmolar fusion medium, fusion ratio, plating density, the use of growth medium without pH indicator, and the use of an irradiated human fibroblast feeder layer. By manipulating these parameters, a high hybrid yield can be achieved with different mouse–human heteromyelomas and Epstein-Barr virus–activated B cells.
Keywords: human hybridoma, human monoclonal antibody, electrofusion, hypoosmolar fusion medium
