Location of the epitope for 7D5, a monoclonal antibody raised against human flavocytochrome b₅₅₈, to the extracellular peptide portion of primate gp91^phox

Flavocytochrome b₅₅₈ is the membrane component of the phagocyte NADPH oxidase, and is a heterodimer composed of gp91^phox and p22^phox subunits. Human flavocytochrome b₅₅₈ 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 23 disease by flow-cytometry. Therefore, were-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 ∼58 kDa unglycosylated gp91^phox protein from solubilized membrane fractions of tunicamycintreated 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 b₅₅₈-deficient chronic granulomatous disease and for elucidating structural characteristics of flavocytochrome b₅₅₈.