Abstract
In the present study, we engineered cDNA encompassing human membrane IgE specific moieties, εm67 (CεmX plus migisR ε), into E. coli expression system. We utilized maltose-binding protein or glutathione-S-transferase as the fusion partner and constructed two recombinant proteins, MBP-εm67-8 and GST-εm67. These two fusion proteins were purified by one-step affinity chromatography: amylose resin for MBP-εm67-8 and glutathione Sepharose 4B gel for GST-εm67. These two fusion proteins were used as antigens to induce anti-εm67 response in Balb/c mice. The anti-εm67 activities of mouse sera were tested by ELISA, western blotting and cell staining of SKO-007 or N6m9 (one transfectant expressing the Fc encompassing cytoplasmic domain of human membrane IgE). The sera from mice immunized with GST-εm67 could recognize recombinant εm67 fusion proteins as well as the human membrane IgE on N6m9 cells. We prepared spleen cells from these mice that exhibited high titer and fused them with NS0 myeloma cells by the PEG method. After screening several thousand hybrids, we found one hybridoma, which secretes antibodies capable of recognizing the denatured εm67, as shown in an immunoblot, but do not recognize εm67 of membrane IgE on B cells, as shown in cell staining assays. This monoclonal antibody implicates that (1) εm67 on human membrane IgE expressing B cells folds into some conformational structure and (2) the peptide recognized by this antibody is not accessible in the native conformation.