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

Abstract

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 (Fc_εR) and IgG (Fc_γR), 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 activity.