Location of the Epitope for 7D5, a Monoclonal Antibody Raised against Human Flavocytochrome b₅₅₈, to the Extracellular Peptide Portion of Primate gp91^phox

Abstract

Flavocytochrome bs« is the membrane component of the phagocyte NADPH oxidase, and is a heterodimer composed of gp9lP hOX and p22P hox subunits. Human flavocytochrome b sss is recognized by monoclonal antibody 7D5 at an unidentified extracellular domain, although our previous study suggested it might recognize p22P hox • 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 gp9lP h0 X. Flow-cytometric analyses showed that 7D5 consistently bound to cells expressing human gp9lP h0 X. 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 gp91PhOX 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 bsss-deficient chronic granulomatous disease and for elucidating structural characteristics of flavocytochrome bsss.

Functional Epitope on Human Neutrophil Flavocytochrome b558

“…This analysis indicated the epitope is not accessible on intact neutrophils (Fig. 1B), compared with the positive control mAb 7D5 which binds an extracellular epitope on human (9) and primate neutrophils (31). However, following permeabilization of neutrophil membranes with 0.05% saponin the staining by mAb NL7 increased significantly, producing a signal that was slightly greater than the positive control mAb 44.1 ( Ref.…”

Helicobacter pyloriDisrupts NADPH Oxidase Targeting in Human Neutrophils to Induce Extracellular Superoxide Release

“…2Ј,7Ј-Dichlorodihydrofluorescein diacetate (DCF) was obtained from Molecular Probes and [ 32 P]orthophosphate (10 mCi/ml) from PerkinElmer Life and Analytical Sciences. Rabbit polyclonal Abs (pAb) to p47 phox and p67 phox and murine mAb to gp91 phox (54.1 and 7D5) and p22 phox (44.1) have been described (15,25). Rabbit anti-lactoferrin (Lf) pAb was obtained from Dr. B. Britigan (University of Iowa, Coralville, IA).…”

“…Low levels of flavocytochrome b 558 are present in the plasma membrane of resting PMNs, and the majority of the protein is located in the membranes of specific and gelatinase granules that are mobilized in response to an activating stimulus (37). We followed up-regulation of flavocytochrome b 558 at the surface of unstimulated, PMA-treated, and Hp-infected PMNs using mAb 7D5, which binds to an extracellular epitope in gp91 phox (25). As shown in Fig.…”

Immunofluorescence and Confocal Microscopy of Neutrophils

“…5 We routinely use the following antibodies for immunofluorescence microscopy of human neutrophils [6][7][8][9][10]. To detect NADPH oxidase components, antibodies specific for p22 phox (mAb 44.1) and gp91 phox (mAb 54.1) [20,21] are now available from Santa Cruz Biotechnology (Santa Cruz, CA); mAb 7D5 detects an epitope of gp91 phox present in mature flavocytochrome b 558 [22] and can be used on intact PMNs to detect protein upregulation at the cell surface [7]; a rabbit mAb specific for human p40 phox (Epitomics, Burlingame, CA); an antibody that specifically detects active Rac (NewEast Biosciences, Malvern, PA); and rabbit antisera from William Nauseef (University of Iowa) specific for p47 phox and p67 phox [6]. Mouse mAbs specific for human CD63, lamp-1, and CD11b are obtained from the University of Iowa Developmental Studies Hybridoma Bank.…”