Affinity Chromatography and SDS-PAGE Studies of Radiolabeled IgE-Binding and IgG-Binding Factors Generated from Human Lymphoblastoid Cell Lines

摘要

Soluble IgE-binding and IgG-binding factors were generated by 18-hour incubation at 4°C of the human B cell lines RPMI 8866 and Daudi. These cells express Fc receptors for IgE (FcεR) and IgG (FcγR), respectively. Binding factors specifically inhibited FcR on both lymphocytes and monocytes, and bound to Ig-Sepharose supports. RPMI 8866 cells and Daudi cells were radiolabeled with 125I by the lactoperoxidase method, and the soluble factors were labeled by the chloramine T method. Affinity chromatography of the soluble factors was performed with IgE-Sepharose, IgG-Sepharose and lentil-lectin-Sepharose followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. The finding of a common 22,000-dalton protein in supernatants with IgE binding, IgG binding, and non-binding activity is discussed in relation to methodological difficulties and the ambiguous results in the literature, as well as the possibility of a complex formation of macromolecules with binding factor activi