Preparation and characterization of a novel agonistic anti-human death receptor 5 monoclonal antibody with apoptosis activity
Objective To prepare anti-hDR5 monoclonal antibody (McAb) with apoptosis activity. Methods To immunize BALB/c mice with rhDR5, and to prepare McAb by hybridoma technique. Tumoricidal anti-human DR5 McAb was screened by MTT assay. McAb was purified by protein G affinity chromatography. IgG subclass was analysed by Sandwich-ELISA. The antigen epitope type of McAb was identified by Western blot and Dot-ELISA. McAb speciality was analysed by indirect-ELISA. The apoptosis of Jurkat cells was detected by flow cytometry with annexin V-FTTC/PI staining. The DNA fragmentation in Jurkat cells was analysed by agarose gel electrophoresis. Results A hybridoma was obtained, which secrets an IgG1 anti-hDR5 McAb with intrinsic cyto-toxicity named as mDRA-6. The epitope type is of confonnation and mDRA-6 specially identificates rDR5. The flow cytometiy analysis showed that phosphatidylserine(PS) highly expressed in Jurkat cell surfaces treated with mDRA-6, and agarose gel electrophoresis exhibited DNA fragmentation in that cells. Conclusion An apoptosis-inducing McAb against hDR5 has been prepared successfully. mDRA-6 is a novel agonistic anti-human DR5 McAb, and it may be a useful tool in tumor therapy by using DR5 as target molecule and in exploring the functional domain of DR5.