Establishment of Monoclonal Antibodies against Human Erythrocyte NADH-Cytochrome b 5 Reductase

Abstract

NADH-cytochrome b₅ reductase (b5R) is a multifunctional redox enzyme, whose deficiency leads to hereditary methemoglobinemia. By using recombinant human red cell b5R as antigen to immunize BALB/c mice and conventional cell fusion, we have established two mouse hybridoma cell lines secreting IgG monoclonal antibodies (MAbs) to b5R. In immunoblotting, the MAbs were shown to react specifically with b5R. They were also found to be capable of capturing b5R activity from b5R solution and normal human hemolysate. It was implied that the binding sites of the MAbs might not be proximal to the active site of the enzyme, but might be in close proximity to each other. The MAbs will be useful in b5R-related investigations.