Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization
Liu, F.T.; Bohn, J.W.; Ferry, E.L.; Yamamoto, H.; Molinaro, C.A.; Sherman, L.A.; Klinman, N.R.; Katz, D.H.
Journal of Immunology 124(6): 2728-2737
1980
ISSN/ISBN: 0022-1767 PMID: 7373045 Document Number: 161886
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 H chain classes, i.e., .mu., .gamma.1 and .gamma.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 MW of 184,000, a total carbohydrate content of 13.3%, and its amino acid composition was determined. The antibody has an association constant with DNP-lysine of 1.4 .times. 108 M-1 at 25.degree. C and 7.1 .times. 107 M-1 at 37.degree. 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 had high specificity and sensitivity. Finally, the isolated hybridoma IgE can mediate antigen (DNP-BSA)-induced release of mediator (serotonin) from rat basophilic leukemia cells.