Unusual polyclonal anti-gp91 peptide antibody interactions with X-linked chronic granulomatous disease-derived human neutrophils are not from compensatory expression of Nox proteins 1, 3, or 4.
To obtain topological information about human phagocyte flavocytochrome b558 (Cytb), rabbit anti-peptide antibodies were raised against synthetic peptides mimicking gp91(phox) regions: 1-9 (MGN), 30-44 (YRV), 150-159 (ESY), 156-166 (ARK), 247-257 (KIS-1, KIS-2). Following affinity purification on immobilized peptide matrices, all antibodies but not prebleed controls recognized purified detergent-solubilized Cytb by enzyme-linked immunosorbent assay (ELISA). Affinity-purified antibodies recognizing KIS, ARK and ESY but not YRV, MGN or prebleed IgG specifically detected gp91(phox) in immunoblot analysis. Antibodies recognizing MGN, ESY, ARK and KIS but not YRV or the prebleed IgG fraction labeled intact normal neutrophils. Surprisingly, all antibodies, with the exception of YRV and pre-immune IgG controls, bound both normal and Cytb-negative neutrophils from the obligate heterozygous mother of a patient with X-linked chronic granulomatous disease (X-CGD) and all neutrophils from another patient lacking the gp91(phox) gene. Further immunochemical examination of membrane fractions derived from nine genetically unrelated patients with X-CGD, using an antibody that recognizes other Nox protein family members, suggests that the unusual reactivity observed does not reflect the compensatory expression of gp91(phox) homologs Nox1, 3 or 4. These results suggest that an unusual surface reactivity exists on neutrophils derived from X-linked chronic granulomatous disease patients that most likely extends to normal neutrophils as well. The study highlights the need for caution in interpreting the binding of rabbit polyclonal antipeptide antibodies to human neutrophils in general and, in the specific case of antibodies directed against Cytb, the need for Cytb-negative controls.
Baniulis
Danas
D
Department of Microbiology, Montana State University, Bozeman, MT, USA.
Nauseef
William M
WM
Burritt
James B
JB
Taylor
Ross M
RM
Heyworth
Paul G
PG
Dinauer
Mary C
MC
Bumelis
Vladas A
VA
Magnusson
Karl-Eric
KE
Jesaitis
Algirdas J
AJ
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D000937:Antigen-Antibody Reactions; D003573:Cytochrome b Group / Q000276:immunology; D000939:Epitopes; D005260:Female; D006105:Granulomatous Disease, Chronic / Q000276:immunology*; D006579:Heterozygote; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000276:immunology*; D008565:Membrane Proteins / Q000032:analysis / Q000096:biosynthesis; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology*; D009504:Neutrophils / Q000276:immunology*; D010455:Peptides / Q000276:immunology
D000906:Antibodies; D003573:Cytochrome b Group; D000939:Epitopes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010455:Peptides; C036888:cytochrome b558; C056558:CYBB protein, human; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C402799:NOX1 protein, human; C498910:NOX4 protein, human; C431570:Nox3 protein, human