Location of the epitope for 7D5, a monoclonal antibody raised against human flavocytochrome b558, to the extracellular peptide portion of primate gp91phox

Flavocytochrome b(558) is the membrane component of the phagocyte NADPH oxidase, and is a heterodimer composed of gp91(phox) and p22(phox) subunits. Human flavocytochrome b(558) is recognized by monoclonal antibody 7D5 at an unidentified extracellular domain, although our previous study suggested it might recognize p22(phox). 7D5 has proven useful in rapid screening of individuals for X-linked chronic granulomatous disease by flow-cytometry. Therefore, we re-evaluated the location of the 7D5 epitope using gene-engineered cell lines expressing hybrid flavocytochromes composed of human and murine subunit homologues. The current study demonstrates that the 7D5 recognizes epitope only of primate gp91(phox). Flow-cytometric analyses showed that 7D5 consistently bound to cells expressing human gp91(phox). In addition, 7D5 immunoprecipitated the similar to 58 kDa unglycosylated gp91(phox) protein from solubilized membrane fractions of tunicamycin-treated PLB-985 granulocytes, indicating that glycans were not required for 7D5 binding. Transgenic COS7 cells expressing human gp91(phox) but not p22(phox) were recognized by 7D5. These results localized the epitope of 7D5 to an extracellular peptide portion of primate gp91(phox) and indicate that the antibody will be useful for monitoring the efficiency of gene therapy in patients with flavocytochrome b(558)-deficient chronic granulomatous disease and for elucidating structural characteristics of flavocytochrome b(558).