Monoclonal antibody 7D5 recognizes the R147 epitope on the gp91^phox , phagocyte flavocytochrome b₅₅₈ large subunit

Human phagocyte flavocytochrome b₅₅₈ (Cyt b), the catalytic center of nicotinamide adenine dinucleotide phosphate oxidase, consists of a heavily glycosylated large subunit (gp91^phox ; Nox2) and a small subunit (p22^phox ). Cyt b is a membrane-spanning complex enzyme. Chronic granulomatous disease (CGD) is predominantly caused by a mutation in the CYBB gene encoding gp91^phox on the X-chromosome. Because the phagocytes of patients with CGD are not able to generate the superoxide anion, these patients are susceptible to severe infections that can be fatal. It has been suggested that the extracellular region of gp91^phox is necessary for and critical to forming the epitope of mAb 7D5 and that 7D5 provides a useful tool for rapid screening of X-linked CGD by FACS. To further elucidate the mAb 7D5 epitope on human gp91^phox , chimeric DNA expressed human and mouse gp91^phox recombinant protein were constructed. The fusion proteins were immunostained for mAb 7D5 and analyzed by FACS and western blot analysis. The ¹⁴³ ELGDRQNES¹⁵¹ region was found to reside at the extracellular surface on human gp91^phox and to be an important epitope for the interaction with mAb 7D5, as analyzed by FACS analysis. In particular, amino acid R147 is a unique epitope on the membrane-associated Cyt b for mAb 7D5. In conclusion, it is proposed that FACS analysis using mAb 7D5 is a valuable tool for early diagnosis of CGD.