Antibody-based spot test for NADH–cytochrome b5 reductase activity for the laboratory diagnosis of congenital methemoglobinemia

Abstract

NADH (reduced Coenzyme I)–cytochrome b5 reductase (b5R) is a multifunctional redox enzyme, whose deficiency causes recessive congenital methemoglobinemia. A novel procedure for the detection of b5R activity in human hemolysates was developed, in which b5R monoclonal antibodies dot-blotted on nitrocellulose membrane was used to capture and enrich b5R from hemolysates, and the captured b5R activity was subsequently visualized with the substrate 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide. Application of this simple method to the detection of b5R activity in the hemolysates from different subjects demonstrated that it was both sensitive and reliable. Our method would be useful for the laboratory diagnosis of congenital methemoglobinemia.