Characterization of Neutrophil NADPH Oxidase Activity Reconstituted in a Cell‐Free Assay Using Specific Monoclonal Antibodies Raised Against Cytochrome b558

Abstract

The immunochemical characterization of NADPH oxidase activity of cytochrome b558 purified from human neutrophils was determined after reconstitution in a cell‐free assay using the native hemoprotein and recombinant purified cytosolic activating factors. The oxidase activity showed a strict dependence on the heme content at each step of the hemoprotein purification process. The immunochemical properties of the reconstituted oxidase made use of monoclonal antibodies raised against membrane‐bound and octyl‐glucoside‐extracted cytochrome b. From nine specific monoclonal antibodies reacting with gp91‐phox cytochrome b558, two were selected, both of which were found to bind to the β subunit of cytochrome b558, and to inhibit superoxide formation in the oxidase reconstituted cell‐free assay. The extent of inhibition was dependent on the phospholipid environment. Neutrophil membrane extracts from X‐linked chronic granulomatous disease patients did not produce O2− in the reconstituted system and did not bind to the antibodies. Copyright © 1995, Wiley Blackwell. All rights reserved