Stability of Specific Immunoglobulin Secretion by EBV-Transformed Lymphoblastoid Cells and Human-Murine Heterohybridomas

Publisher Website
Google Scholar
Abstract
We have examined variables leading to the generation of stable, antigen-specific, human immunoglobulin-secreting cell lines. Peripheral blood B lymphocytes enriched for Thomsen-Friedenreich antigen (T antigen)-specific cells were transformed with Epstein-Barr virus. Lymphoblastoid cells (LC) reactive with T antigen were either expanded without cloning or cloned at limiting dilution and then fused with murine 653 cells. Uncloned LCs from three transformations secreting polyclonal anti-T antibody (7-18 μg/ml/106 cells/24 hr total immunoglobulin) were subcultured at 100 cells/well, and T antigen-reactive cultures pooled. These cultures quickly lost specific antibody secretion, presumably due to overgrowth by clones of unknown specificity. T antigen-reactive LCs that were cloned three times at limiting dilution secreted 0.2 - 6.1 μg/ml/106 cells/24 hr but died or stopped secreting specific immunoglobulin after 77 to 155 days in culture. Pooling T antigen-reactive clones after each cloning step did not increase the long term stability compared to unpooled clones (p - 0.2). Fusions between cloned LCs and 653 cells failed to yield viable hybrids in nine of ten attempts with seven different LC lines. In contrast, fusion of uncloned LCs and 653 cells resulted in the generation of viable immunoglobulin-secreting heterohybrids in 22 of 24 fusions. The heterohybridomas produced from fusion of uncloned T antigen-reactive cultures with 653 cells secreted significantly more antibody (frequency of cell lines secreting greater than 2 μg/ml/106cells/24 hr, p<0.01) and higher titers of antibody (frequency of cell lines secreting greater than four hemagglutination units of T antigen-specific antibody, p<0.03) than cloned lymphoblastoid cells. The hybrids maintained specific immunoglobulin secretion for longer in culture than either cloned or uncloned lymphoblastoid cell lines (p<0.001).