A novel epitope (pentapeptide) in the human hemoglobin beta chain

We have cloned 16 monoclonal antibodies by immunizing mice with human hemoglobin for the purpose of analyzing epitopes in human hemoglobin. By using one, SU-115, which is specific for the beta chain, an epitope in the beta chain reacting with this monoclonal antibody was investigated, and a pentapeptide was identified as a novel epitope. After digestion of the beta chain by lysylendopeptidase, the antigenicity of degradation products was examined. An antigenic fragment against SU-115 was found to be a peptide corresponding to residues 96-120 of the beta chain by amino acid analysis of its N-terminal region. Several peptides involved in the region of beta96-120 were synthesized to be examined for their reactivity to SU-115 using dot-blot analysis and a resonant mirror detection method, and a pentapeptide (N108VLVC) was determined as a major sequence of an epitope. By injecting this pentapeptide into a mouse, an antibody reacting human hemoglobin (alpha2beta2) in the same order of strength as SU-115, was obtained. The pentapeptide described in this paper seems to be the minimum size as a major sequence of the actual epitope in human hemoglobin so far reported, and in the primary structure of this region (108-112) there is a difference of three amino acids between human and mouse hemoglobin.