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 cytoc hrome b(558) purified from human neutrophils was determined after reco nstitution in a cell-free assay using the native hemoprotein and recom binant purified cytosolic activating factors. The oxidase activity sho wed a strict dependence on the heme content at each step of the hemopr otein purification process. The immunochemical properties of the recon stituted oxidase made use of monoclonal antibodies raised against memb rane-bound and oclyl-glucoside-extracted cytochrome b. From nine speci fic monoclonal antibodies reacting with gp91-phox cytochrome b(558), t wo were selected, both of which were found to bind to the beta subunit of cytochrome b(558) and to inhibit superoxide formation in the oxida se reconstituted cell-free assay. The extent of inhibition was depende nt 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.