Characterization of human hybridomas secreting antibody to tetanus toxoid.

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We have selected a thioguanine-resistant lymphoblastoid cell line (LTR228) that forms human-human hybrids with high efficiency. Fusions with peripheral B cells consistently yield one colony per 10(5) cells plated. To produce antitetanus monoclonal antibodies, we withdrew blood from persons who had recently received booster injections of tetanus toxoid. T cells were separated from peripheral mononuclear cells by 2-aminoethylisothiouronium bromide-induced rosette formation, given 1,500 rads (1 rad = 0.01 gray), and cultured in a 1:1 ratio with nonrosetting cells. After 3 days of pokeweed mitogen stimulation, heterokaryons were produced by a plate-fusion technique and cultured in Iscove's Dulbecco's minimal essential medium for 24 hr prior to hybrid selection. Colonies appeared after 10-14 days in hypoxanthine/azaserine supplemented medium. A direct binding enzyme-linked immunosorbent assay with specific tetanus toxoid inhibition identified positive wells. The hybridomas were cloned twice in soft agarose and by limiting dilution. The subcloned hybridomas double every 26 hr (vs. every 16 hr for LTR228) and produce 1-5 micrograms of specific IgG, kappa antibody per 10(6) cells per ml per 24 hr. All subclones (almost 200) continue to secrete antibody after 11 months of continuous culture. Twelve representative subclones have near tetraploid amounts of DNA. From hybridomas grown in 5-liter spinner flasks, milligram quantities of the IgG, kappa antibody were purified by staphylococcus protein A affinity chromatography. Specific antibody from hybridoma cultures protected mice injected with 1,000 times the LD50 of tetanus toxin. Our cell line and associated techniques should permit the production of therapeutically important human monoclonal antibodies.

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