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.

Chronic granulomatous disease: Clinical, functional, molecular, and genetic studies. The Israeli experience with 84 patients

“…Neutrophil lysate (20 μg) was electrophoresed in 10% (w/v) SDS‐polyacrylamide gel and transferred to a nylon membrane. The membrane was incubated with mouse monoclonal antibodies against gp91 phox or p22 phox or with specific rabbit polyclonal antisera to p47 phox or p67 phox . Alkaline phosphatase‐conjugated anti‐mouse or anti‐rabbit immunoglobulin was used to detect antibody binding to the blot.…”

Characterization of ThOX Proteins as Components of the Thyroid H2O2-Generating System

“…ThOX proteins were detected using the rabbit polyclonal antibody raised against the intracellular part of ThOX1 as previously described [12]. TPO and p22 Phox proteins were detected by monoclonal antibodies MoAb 47 and MoAb 448, respectively, received from Dr. J. Ruf [20] and Dr. D. Roos [21]. The immune complexes were detected using a horseradish peroxidase-coupled anti-mouse antibody according to the ECL method (NEN Life Science Products).…”

A novel point mutation in the CYBB gene promoter leading to a rare X minus chronic granulomatous disease variant — Impact on the microbicidal activity of neutrophils

“…Neutrophil proteins were solubilised in 1% Triton X-100, separated by SDS-PAGE in 10% (w/v) acrylamide gel with a 5% (w/v) stacking gel [29], electrotransferred to nitrocellulose [30] and immunodetected using the monoclonal antibodies 449 and 48 directed against p22phox and gp91phox, respectively [31] and by polyclonal antibodies directed against p47phox and p67phox [32]. Electrophoretic mobility shift assays were performed in a 20-µl total volume with 10 µg of nuclear protein extract in 10 mM Tris, 50 mM KCl, 1 mM DTT (pH 7.5), 50 ng/µL Poly (dI- Purified genomic DNA was amplified using a forward primer P1…”