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.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Schmidt E,Leinfelder U,Gessner P,Zillikens D,Bröcker EB,Zimmermann Udoi
10.1016/s0022-1759(01)00431-8keywords:
subject
Has Abstractpub_date
2001-09-01 00:00:00pages
93-102issue
1-2eissn
0022-1759issn
1872-7905pii
S0022175901004318journal_volume
255pub_type
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