An efficient procedure for the generation of human monoclonal antibodies based on activation of human B lymphocytes by a murine thymoma cell line.
A new, efficient procedure for the generation of human monoclonal antibodies has been developed. The procedure is based on the activation of human B cells in microwells by murine thymoma EL4B5 cells. This mode of B cell stimulation leads to proliferation of at least one per eight of human B cells and to a high rate of antibody production. Subsequently, supernatants of the microwells are screened by ELISA for the presence of antibody of the desired specificity and B cells from selected wells are hybridized by electroporation. To optimize the procedure, the kinetics of the B cell expansion induced by EL4B5 cells were analysed. Counting and phenotyping of cultured cells at different time points indicated that the peak of B cell expansion occurred at day 5 for tonsil B cells (16-fold increase) and at day 7 for peripheral blood B cells (20-fold increase). The B cells did not merely proliferate but also differentiated, as indicated by loss of CD20 expression and increase of CD38 expression. At the peak of B cell expansion, B cells could be hybridized efficiently with myeloma cells. The majority of the resultant hybridomas secreted human immunoglobulin. The efficiency of the procedure is exemplified by the generation of hybridomas secreting human IgG against Haemophilus influenzae from limited numbers of either human tonsil B lymphocytes or peripheral blood B lymphocytes.
Kwekkeboom
J
J
Laboratory of Cell Biology and Histology, University of Amsterdam, Academic Medical Center, Netherlands.
van Oosten
M
M
de Boer
M
M
van Alphen
L
L
Mevissen
M L
ML
Lindhout
E
E
Tager
J M
JM
de Groot
C
C
D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D001402:B-Lymphocytes / Q000276:immunology*; D002454:Cell Differentiation; D002455:Cell Division; D002648:Child; D004797:Enzyme-Linked Immunosorbent Assay; D006193:Haemophilus influenzae / Q000276:immunology; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D008213:Lymphocyte Activation; D051379:Mice; D013945:Thymoma / Q000276:immunology*; D013953:Thymus Neoplasms / Q000276:immunology*; D014407:Tumor Cells, Cultured
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G