Monoclonal antibody CL5 recognizes the amino terminal domain of human phagocyte flavocytochrome b 558 large subunit, gp91 phox Flavocytochrome b 558 (Cytb) is the central com- ponent of the b-nicotinamide adenine dinucleo- tidephosphate, reduced (NADPH)-oxidase, a multi-subunit enzyme system that produces super- oxide (O 2 ) ) (1–3). The oxidase is an important element in host defense by phagocytic cells. Dysfunction of the NADPH-oxidase causes chro- nic granulomatous disease (CGD), an inherited disorder characterized by severe recurrent infec- tions (4). Recently, structural homologs of the Cytb large subunit gp91 phox have been identified in a variety of tissues and other organisms suggesting alternate functions for superoxide and Cytb-like proteins (reviewed in 5, 6). The NADPH oxidase complex consists of mem- brane-bound Cytb, the low molecular weight G proteins Rap1A and Rac2, and the cytoplasmic proteins p47 phox , p67 phox , and p40 phox (7). Activa- tion of the NADPH-oxidase by a variety of agents requires translocation of cytosolic oxidase compo- nents to the plasma membrane where they associate with Cytb to form a functional oxidase (reviewed in Baniulis D, Burritt JB, Taylor RM, Dinauer MC, Heyworth PG, Parkos CA, Magnusson K-E, Jesaitis AJ. Monoclonal antibody CL5 recognizes the amino terminal domain of human phagocyte flavocytochrome b 558 large subunit, gp91 phox . Eur J Haematol 2005: 74: 337–347. Ó Blackwell Munksgaard 2005. Abstract: Human phagocyte flavocytochrome b 558 (Cytb) is a heterodimeric integral membrane protein that serves as the electron transferase of the b-nicotinamide adenine dinucleotidephosphate, reduced (NADPH)-oxidase, an enzyme complex important in the host defense function of phagocytic cells. In this study, we report the char- acterization of monoclonal antibody (mAb) CL5 that is specific for the large subunit, gp91 phox , of the oxidase protein. This antibody recognizes gp91 phox by immunoblot analysis of membrane extracts and samples of the immunopurified gp91 phox /p22 phox heterodimer, prepared on anti-p22 phox affinity matrices. Phage display analysis confirmed this specificity, indicating that the CL5 epitope contains the region 135- DPYSVALSELGDR of gp91 phox . The antibody was used to probe for the presence of gp91 phox in membrane preparations from neutrophils of patients with nine genetically distinct forms of X-linked chronic gra- nulomatous disease (CGD). The causative mutations included missense errors as well as nonsense errors that result in premature termination of gp91 phox synthesis. Analysis of the CGD samples by immunoblotting indicated that CL5 recognizes only the full-length wild-type and two missense mutations, consistent with the absence of stable short gp91 phox peptide expression in CGD neutrophils. Interestingly, CL5 was also shown to be cross-reactive with cytosolic and membrane-bound gelsolin, identified by purification, mass spectrometry and immunoblot analysis. CL5 probably cross-reacts with the sequence 771-DPLDRAMAEL in the C-terminus of gelsolin. We conclude that mAb CL5 is a useful probe for detection of full length and possibly truncated N-terminal fragments of gp91 phox from membranes of Cytb-producing cells. Danas Baniulis 1 , James B. Burritt 1 , Ross M. Taylor 1 , Mary C. Dinauer 2 , Paul G. Heyworth 3 *, Charles A. Parkos 4 , Karl-Eric Magnusson 5 , Algirdas J. Jesaitis 1 1 Department of Microbiology, Montana State University, Bozeman, MT, USA; 2 Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA; 3 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA; 4 Department of Pathology, Emory University, Atlanta, GA, USA; 5 Division of Medical Microbiology, Department of Health and Environment, School of Medicine, Linkçping University, Linkçping, Sweden Key words: monoclonal antibody; epitope mapping; gp91 phox ; flavocytochrome b 558 ; CGD; gelsolin Correspondence: Algirdas J. Jesaitis, Department of Microbiology, Montana State University, Bozeman, MT 59717, USA Tel: +406-994-4811 Fax: +406-994-4926 e-mail: umbaj@montana.edu *Present address: DNAX Research, Inc., Palo Alto, CA, USA Accepted for publication 24 September 2004 Eur J Haematol 2005: 74: 337–347 All rights reserved Copyright Ó Blackwell Munksgaard 2005 EUROPEAN JOURNAL OF HAEMATOLOGY 337