Preparation of membrane binding monoclonal antibody anti rat FcεRIα and dectection of its activity

Objective:To induce the mast cell apoptosis by specifically targeting the monoclonal antibody (mAb) against FceRIα. Methods: Splenocytes of BALB/c mice immunized with rat basophilic leukemia cells line RBL-2H3 were fused with myeloma cells line SP2/0. ELISA assay was used to screen antibody secreting from mouse sera and fused cells. Hybridoma cell lines were cloned by limiting dilution. The character and bioactivity of the antibodies were measured by immune electrophoresis on SDS-PAGE gel,immunodifusion,competitive inhibition and histamine release. The antigen component was verified by analyzing the autoradiography of precipitation of immune complexes of 125I-labeled RBL-2H3 and mAbs. Results: Two monoclonal antibody cell lines ER-E5. 3 and ER-C7. 4 were acquired. The antibodies were specifically against rat FceRIα and all belonged to IgGl subtype. The titrations of antibodies in supernatant and mouse ascites were higher than 105. Flow cytometry (FCM) showed that the binding rate of antibodies to RBL-2H3 cells was over 95% but no cross-linked activity to rat thymus and peripheral blood lymphocytes (PBL) was detected. The antibodies prevented IgE bind to RBL-2H3 and the histamine release was equivalent to that of DNP-lgE triggered RBL-2H3. Fab prepared by papain-digestion of the whole antibody showed the same activity as whole antibody in self-binding and inhibiting IgE binding to RBL-2H3,but lost the ability to trigger histamine release. Immunoprecipitation and autoradiography demonstrated that the antibody was directly against membrane FceRIα-subunit. Conclusion: Two hybridoma clones ER-E5. 3 and ER-C7. 4 have been obtained. The titration and activity of the 2 antibodies all meet the requirement for experimental study and the antigen can be specifically recognized by FceRα.