Characterization of two monoclonal antibodies against cytochrome b558 of human neutrophils

Abstract

Monoclonal antibodies (MoAbs) were raised against cytochrome b558, a membrane-bound component of the NADPH:O2 oxidoreductase in human neutrophils. This cytochrome consists of a low-molecular-weight (low- mol-wt) subunit of 22 to 23 Kd, probably encoded by an autosomal gene, and a high-mol-wt subunit of 75 to 90 Kd, encoded on the X-chromosome. MoAb 449 reacts with the low-mol-wt subunit and MoAb 48 with the high- mol-wt subunit on Western blots of purified cytochrome b558 and on blots of whole neutrophil extracts. In extracts of neutrophils from patients with chronic granulomatous disease (CGD) in which cytochrome b558 is not detectable by spectrophotometric methods, the low-mol-wt subunit is present, albeit in a much smaller amount. The high-mol-wt subunit is not detected by MoAb 48 in neutrophils of patients with X- linked CGD and in neutrophils of patients with the autosomal, cytochrome-b558-negative form of the disease. These results can be explained by a marked instability of these subunits when the synthesis of either of the two is disturbed. In differentiated HL-60 cells, the high-mol-wt subunit appears to be present in a different form. Cloning of the low-mol-wt subunit with the help of MoAb 449 suggests the presence of a heme-binding site on this subunit. By comparison of the binding characteristics of MoAb 449 to intact and permeabilized neutrophils with those of MoAb 7D5, recently isolated by Nakamura et al (Blood 69:1404, 1987), the low-mol-wt subunit was established as a transmembrane protein.

Skewing of X‐chromosome inactivation in three generations of carriers with X‐linked chronic granulomatous disease within one family

“…To identify the affected subunit of the patients' NADPH oxidase, the expression of cytochrome b 558 was measured by incubating the neutrophils with monoclonal anticytochrome b 558 antibodies (MoAb 7D5 for gp91-phox [gift from Dr M. Nakamura, Nagasaki, Japan] and MoAb 449 for p22phox [from Sanquin, Amsterdam, the Netherlands]). These MoAbs are specific for these phox proteins [6,15]. Rabbit polyclonal antibodies for cytoplasmic p47-phox and p67-phox proteins were from Upstate Serological Company (Waltham, MA).…”

“…Both physical studies and peptide sequencing of purified flavocytochrome b 558 indicate that it is a heterodimer with a stoichiometry of 1 : 1 gp91-phox and p22phox polypeptides [5]. Heterodimer formation appears to be important for stable expression of each subunit in the NADPH oxidase, because virtually all patients with flavocytochrome b 558 mutations lack both gp91-phox and p22-phox polypeptides, regardless of which subunit is affected by the genetic defect [6,7].…”

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

“…Immunoblotting of cytochrome b. Purified neutrophils treated with 3 mM iPrJ-F were lysed as described (Verhoeven et al, 1989). Briefly, the neutrophils (10' cells) were suspended in a lysis buffer containing 1 % (mass/vol.)…”

Monoclonal antibody CL5 recognizes the amino terminal domain of human phagocyte flavocytochrome b558 large subunit, gp91phox

“…Several monoclonal antibodies recognizing the large subunit of Cytb have been reported. Several such antibodies have been characterized, namely 54.1 (30) and NL7 (31) produced by Jesaitis et al, antibody 7D5 produced by Nakamura et al (32,33,34) and antibodies 48 and 449 produced by Roos and colleagues (35,38). These antibodies have been used extensively to provide information about the expression of Cytb, including CGD studies (35)(36)(37).…”

“…Several such antibodies have been characterized, namely 54.1 (30) and NL7 (31) produced by Jesaitis et al, antibody 7D5 produced by Nakamura et al (32,33,34) and antibodies 48 and 449 produced by Roos and colleagues (35,38). These antibodies have been used extensively to provide information about the expression of Cytb, including CGD studies (35)(36)(37). The use of these mAb has also helped to advance our understanding of Cytb structure and function (14,30,31,34,38).…”