CHARACTERIZATION OF NEUTROPHIL NADPH OXIDASE ACTIVITY RECONSTITUTED IN A CELL-FREE ASSAY USING SPECIFIC MONOCLONAL-ANTIBODIES RAISED AGAINST CYTOCHROME B(558)

The immunochemical characterization of NADPH oxidase activity of cytochrome b(558) 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 oclyl-glucoside-extracted cytochrome b. From nine specific monoclonal antibodies reacting with gp91-phox cytochrome b(558), two were selected, both of which were found to bind to the beta subunit of cytochrome b(558) 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 O-2(-) in the reconstituted system and did not bind to the antibodies.