Monoclonal dinitrophenyl-specific murine IgE antibody: Preparation, isolation, and characterization

A murine hybridoma secreting monoclonal IgE antibodies of anti-2,4-dinitrophenyl (DNP) specificity was generated by fusion of SP2/0 tumor cells and spleen cells from DNP-Ascaris-hyperimmunized mice. Hybridomas secreting anti-DNP antibodies of other heavy chain classes, i.e., μ, γ ₁ and γ(2b), were also obtained from the same fusion experiment. Large quantities of IgE antibodies were obtained from ascites of mice in which the IgE-secreting hybridoma was propagated in vivo. The IgE antibodies were isolated by precipitation with ammonium sulfate followed by affinity chromatography on DNP-bovine serum albumin (BSA)-Sepharose-4B and further purified by ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The isolated IgE has an approximate m.w. of 184,000, a total carbohydrate content of 13.3%, and its amino acid composition has been determined. The antibody has an association constant with DNP-lysine of 1.4 x 10 ⁸ M ^-1 at 25°C and 10 ^{7 7} M ^-1 at 37°C. Rabbit and goat antibodies against the hybridoma IgE were prepared and the antisera were made specific for IgE by adsorption on normal mouse serum protein-Sepharose-4B. Solid phase radioimmunoassays for measuring murine antigen-specific and total IgE were developed and were shown to have high specificity and sensitivity. Finally, the isolated hybridoma IgE can mediate antigen (DNP-BSA)-induced release of mediator (serotonin) from rat basophilic leukemia cells.