A Novel Epitope (Pentapeptide) in the Human Hemoglobin β Chain

Abstract

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 β chain, an epitope in the β chain reacting with this monoclonal antibody was investigated, and a pentapeptide was identified as a novel epitope. After digestion of the β 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 β chain by amino acid analysis of its N-terminal region. Several peptides involved in the region of β96–120 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 (α₂β₂) 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.