Production of antigen-specific human monoclonal antibodies from in vitro-primed human splenocytes.
We have developed culture conditions for human lymphocytes that support primary in vitro immune responses to protein Ag of either human or nonhuman origin. We now show that these primed B cells can be efficiently immortalized by fusion with a heterohybrid fusion partner to generate human, Ag-specific IgM or IgG antibody-producing heterohybridomas at a rate of 17 to 50 hybrids/10(6) lymphocytes fused. Approximately 50% of the Ig-secreting clones were stable with respect to Ig secretion. Levels of secretion attained with terminal cultures ranged from less than 1 to 100 micrograms/ml. Fusions of cells between 2 and 5 days after initiation of in vitro exposure to Ag produced more Ag-reactive and Ag-specific antibodies than fusions at 1 day or fusions performed after 5 days. Ag-reactive hybrids could be isolated at frequencies of 3 to 10%, depending on antigenicity of the immunogen. Foreign proteins, horse spleen ferritin, and a murine monoclonal Ig, induced higher percentages of Ag-reactive mAb than immunization with the human-derived ferritin. Ag-reactive IgG mAb were produced at relatively high frequency, depending on immunization conditions and the nature of the Ag. The strategy for identification of the best hybrids included early elimination of unstable hybridomas and of hybridomas producing broadly cross-reactive antibody, followed by evaluation of units of Ag reactivity/micrograms Ig. Ferritin-specific mAb selected according to these criteria showed immunocytochemical reactivity with ferritin-containing tissues and apparent affinities in the range of 10(7) to 10(8)/mol.
Boerner
P
P
University of California, San Diego, La Jolla 92093.
Lafond
R
R
Lu
W Z
WZ
Brams
P
P
Royston
I
I
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000915:Antibody Affinity; D002478:Cells, Cultured; D005293:Ferritins / Q000276:immunology; D006736:Horses; D006801:Humans; D006825:Hybridomas / Q000166:cytology*; D066298:In Vitro Techniques; D013045:Species Specificity; D013154:Spleen / Q000166:cytology* / Q000276:immunology
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D005293:Ferritins