Objective: To prepare and characterize monoclonal antibodies (mAb) against recombinant human receptor for advanced glycation end product (rhRAGE).
Methods: BALB/c mice were immunized with recombinant extracellular domain amino acid 23-342 of human advanced glycation end product (RAGE), and hybridoma was generated with mouse spleen cells and myeloma NS-1 cells. After three fusions and cloning, two hybridoma cell lines secreting monoclonal antibodies to RAGE were obtained and monoclonal antibodies were purified from the ascites followed by characterization with indirect enzyme-linked immunosorbent assay (ELISA), flow cytometry and Western blotting.
Results and conclusion: Two hybridoma cell lines secreting anti-RAGE mAb were established and designated as B2.2 and E10, respectively. Both of mAb B2.2 and E10 belonged to IgG2b isotype and could bind to recombinant RAGE and natural RAGE expressed on THP-1 cells. In addition, B2.2 and E10 could recognize different epitopes of RAGE, which were conformed to be capable of detecting soluble recombinant RAGE in sandwich ELISA. These two mAbs against different epitopes of rhRAGE would be useful for study of glycation end product and RAGE-related diseases.