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Unusual polyclonal anti-gp91phox peptide antibody interactions with X-linked chronic granulomatous disease-derived human neutrophils are not from compensatory expression of Nox proteins 1, 3, or 4

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Baniulis D, Nauseef WM, Burritt JB, Taylor RM, Heyworth PG, Dinauer MC, Bumelis VA, Magnusson K-E, Jesaitis AJ. Unusual polyclonal anti-gp91phox peptide antibody interactions with X-linked chronic granulomatous disease-derived human neutrophils are not from compensatory expression of Nox proteins 1, 3, or 4.

Eur J Haematol 2005: 74: 241–249. © Blackwell Munksgaard 2005. Abstract: 

To obtain topological information about human phagocyte flavocytochrome b558 (Cytb), rabbit anti-peptide antibodies were raised against synthetic peptides mimicking gp91phox 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 gp91phox 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 gp91phox 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 gp91phox 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.
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Keywords: X-CGD; flavocytochrome b558; gp91phox; polyclonal antibodies; transmembrane topology

Document Type: Research Article

Affiliations: 1: Department of Microbiology, Montana State University, Bozeman, MT, USA 2: Inflammation Program, Department of Medicine, Veterans Affairs Medical Center and University of Iowa, Iowa City, IA, USA 3: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA 4: Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA 5: Laboratory of Bioinformatics, Vilnius Technical University of Gediminas, Vilnius, Lithuania 6: Division of Medical Microbiology, Department of Health and Environment, School of Medicine, Linköping University, Linköping, Sweden

Publication date: March 1, 2005

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