Purification and partial biochemical characterization of IgE-binding factors secreted by a human B lymphoblastoid cell line.
IgE-binding factors (IgE-BFs) were purified from the culture supernatant of RPMI-8866 cells, a human lymphoblastoid B-cell line expressing IgE receptors. The material, purified by affinity-chromatography on immunoadsorbents coupled to IgE or to monoclonal antibody against IgE receptor, was comprised of two major components with apparent molecular weight (MW) of 25,000-27,000 and 12,000, as determined by SDS-PAGE and silver staining. Only the 25,000-27,000 MW molecules were identified as IgE-BFs, as demonstrated by their reactivity with MabER in the Western blot and the immunoprecipitation assays, and their ability to inhibit rosette formation of U937 cells with IgE- but not with IgG-coated erythrocytes. IgE-BFs were purified to homogeneity by combining affinity-chromatography and either DEAE-ion exchange or reverse-phase chromatography on an HPLC system. Chromatofocusing analysis demonstrated the microheterogeneity of IgE-BFs that were comprised of molecules with isoelectric points ranging from 5.0 to 4.4. IgE-BFs were sensitive to treatment with O-glycosidase but not with N-glycanase. These molecules were resistant to heat and to pH ranging from 2 to 9; their immunoreactivity was lost after treatment with trypsin and pepsin. Papain digestion of purified IgE-BFs generated 14,000-16,000 MW molecules that were still binding to IgE and to MabER.
D001402:B-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004798:Enzymes / Q000494:pharmacology; D006801:Humans; D007526:Isoelectric Point; D008222:Lymphokines / Q000302:isolation & purification*; D008228:Lymphoma, Non-Hodgkin / Q000276:immunology; D029584:Prostatic Secretory Proteins; D013491:Suppressor Factors, Immunologic / Q000302:isolation & purification*
D004798:Enzymes; D008222:Lymphokines; D029584:Prostatic Secretory Proteins; D013491:Suppressor Factors, Immunologic; C043076:beta-microseminoprotein; C013059:immunoglobulin-binding factors