title: CD19+ B lymphocytes are the major source of human antibody-secreting hybridomas generated by electrofusion.
abstract: 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.
authors.affiliation: Department of Biotechnology, Biocenter, University of Würzburg, Am Hubland, 97074, Würzburg, Germany.
authors.forename: E
authors.initials: E
authors.lastname: Schmidt
authors.forename: U
authors.initials: U
authors.lastname: Leinfelder
authors.forename: P
authors.initials: P
authors.lastname: Gessner
authors.forename: D
authors.initials: D
authors.lastname: Zillikens
authors.forename: E B
authors.initials: EB
authors.lastname: Bröcker
authors.forename: U
authors.initials: U
authors.lastname: Zimmermann
mesh_terms: 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
chemical_list: D000911:Antibodies, Monoclonal; D018941:Antigens, CD19; D010835:Phytohemagglutinins; D010940:Plant Proteins; C101135:phytohemagglutinin L protein, Phaseolus vulgaris