Preparation and identification of monoclonal antibodies against humanin

Abstract

To generate and characterize a monoclonal antibody (mAb) against humanin (HN), BALB/c mice were immunized with the purified pet-44a-HN in adjuvant and their splenic lymphocytes were fused with myeloma SP2/0 cells. The hybridoma cell lines were screened for HN-specific antibodies by indirect enzyme-linked immunosorbent assay (ELISA), and anti-HN mAb-producing hybridoma clones were obtained using a limiting dilution assay. The specificity and affinity of the antibodies were characterized by western blot assays and indirect ELISA. Following fusion, screening and cloning, four hybridoma clones were obtained, and the clone 5A8H3 was demonstrated to stably produce anti-HN IgG2a. Further characterization of 5A8H3 revealed that this mAb specifically recognized HN, the fusion proteins of pet- 44a-HN protein and pGEMEX-1-HN, but not control (Escherichia coli proteins). This mAb interacted with HN at an affinity constant (Ka) of 2.0 × 10⁸ M^–1 The HN-specific IgG2a mAb was successfully generated. It interacted with HN specifically and sensitively, providing a valuable tool for further study of the biological functions of HN.

Key words: Humanin, monoclonal antibodies, characterization