CD19+ B lymphocytes are the major source of human antibody-secreting hybridomas generated by electrofusion.
Human monoclonal antibodies may be generated by electrofusion of human B lymphocytes with a human/mouse heteromyeloma line. In addition to a fusion protocol optimised for the fusion partners, the activation of B lymphocytes is crucial for fusion and hybrid efficiency. In this study, we initially treated peripheral blood mononuclear cells (PBMC) from normal blood donors with a large panel of known stimulants and determined the yield of human antibody-secreting hybridomas after electrofusion with the heteromyeloma cell line H73C11; 3- to 5-day incubation with phytohaemagglutinin L (PHA-L) resulted in the highest number of secreting hybrids. In a second set of experiments, PBMC were depleted from various cell populations, including CD14+ monocytes, CD8+ T lymphocytes, and CD2+ T cells, respectively. Undepleted PBMC stimulated with PHA-L were shown to give rise to the highest number of secreting hybridomas when subjected to electrofusion, whereas depletion of CD2+ T lymphocytes greatly reduced the yield. In a final set of experiments, CD19+ B lymphocytes were identified as the major source of secreting hybridomas. For optimal fusion efficiency, CD19+ B cells were shown to require direct physical contact with other cell populations, most probably T lymphocytes, during the stimulation process. Our data highlight the importance of an adequate stimulation prior to electrofusion and may be helpful to further facilitate the development of human monoclonal antibodies.
Schmidt
E
E
Department of Biotechnology, Biocenter, University of Würzburg, Am Hubland, 97074, Würzburg, Germany.
Leinfelder
U
U
Gessner
P
P
Zillikens
D
D
Bröcker
E B
EB
Zimmermann
U
U
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D018941:Antigens, CD19 / Q000302:isolation & purification*; D016175:B-Lymphocyte Subsets / Q000378:metabolism*; D002459:Cell Fusion / Q000379:methods*; D006801:Humans; D006825:Hybridomas / Q000378:metabolism*; D008213:Lymphocyte Activation; D008297:Male; D051379:Mice; D010835:Phytohemagglutinins / Q000494:pharmacology; D010940:Plant Proteins
D000911:Antibodies, Monoclonal; D018941:Antigens, CD19; D010835:Phytohemagglutinins; D010940:Plant Proteins; C101135:phytohemagglutinin L protein, Phaseolus vulgaris