Tissue Antigens ISSN 0001-2815 Epitope mapping of mAbs to denatured human testicular ACE (CD143) I. V. Balyasnikova 1, *, R. Metzger 2 , F. E. Franke 3 , N. Conrad 4 , H. Towbin 5,† , D. E. Schwartz 1 , E. D. Sturrock 4 & S. M. Danilov 1 1 Department of Anesthesiology, University of Illinois at Chicago, Chicago, IL, USA 2 Department of Pediatric Surgery, University of Leipzig, Leipzig, Germany 3 Institute of Pathology, University of Giessen, Giessen, Germany 4 Division of Medical Biochemistry, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, South Africa 5 Novartis Pharma AG, Basel, Switzerland Key words Angiotensin I-converting enzyme; CD143; immunohistochemistry; Western blotting Correspondence Sergei M. Danilov, MD, PhD Anesthesiology Research Center University of Illinois at Chicago 1819 W. Polk St. (M/C 519) Chicago, IL 60612 USA Tel: 11 312 413 7526 Fax: 11 312 996 9680 e-mail: danilov@uic.edu Received 9 April 2008; revised 23 June 2008; accepted 9 July 2008 doi: 10.1111/j.1399-0039.2008.01112.x Abstract Angiotensin I-converting enzyme (ACE; CD143) has two homologous enzymat- ically active domains (N and C) and plays a crucial role in blood pressure regulation and vascular remodeling. A wide spectrum of monoclonal antibodies (mAbs) to different epitopes on the N and C domains of human ACE have been used to study different aspects of ACE biology. In this study, we characterized a set of nine mAbs, developed against the C domain of human ACE, which recognize the denatured forms of ACE and thus are suitable for the detection and quantification of somatic ACE (sACE) and testicular ACE (tACE) using Western blotting and immunohis- tochemistry on paraffin-embedded human tissues. The epitopes for these mAbs were defined using species cross-reactivity, phage display library screening, Western blotting and ACE mutagenesis. Most of the mAbs recognized common/overlapping region(s) on both somatic and testicular forms of human ACE, whereas mAb 4E10 was relatively specific for the testicular isoform and mAb 5B9 mainly recognized the glycan attached to Asn 731. This set of mAbs is useful for identifying even subtle changes in human ACE conformation because of denaturation. These mAbs are also sensitive tools for the detection of human sACE and tACE in biological fluids and tissues using proteomic approaches. Their high reactivity in paraffin-embedded tissues provides opportunities to study changes in the pattern of ACE expression and glycosylation (particularly with mAb 5B9) in different tissues and cells. Introduction Angiotensin I-converting enzyme (ACE; CD143) is zinc metallopeptidase that converts angiotensin I to angiotensin II, a potent vasopressor, and degrades bradykinin, a potent vasodilator. Thus, this enzyme plays a key role in the regulation of blood pressure and the development of vascular pathology and remodeling. ACE is constitutively expressed on the surface of endothelial cells, different absorptive epithelial and neuroepithelial cells (1–3), and cells of the immune system such as activated macrophages, dendritic cells (4) and endothelial progenitor cells (Danilov SM (University of Illinois, Chicago, IL), Urbich C, Dimmeler S (University of Frankfurt, Germany) and Yoder M (Indiana University, Indianapolis, IN), unpublished observations). Recently, ACE was defined as a new marker of hematopoietic stem cells (5, 6). Somatic ACE (sACE) contains two homologous N- and C-terminal domains (7). A short testis-specific isoform, testicular ACE (tACE), solely expressed in germ cells during spermatogenesis, is comprised of only the C-terminal domain and a unique 36- residue sequence at its N-terminus (8, 9). Disruption of the *Present address: Department of Surgery, University of Chicago, Chicago, IL, USA † Present address: Institute of Pharmaceutical Sciences, Zurich, Switzerland 354 ª 2008 The Authors Journal compilation ª 2008 Blackwell Munksgaard  Tissue Antigens 72, 354–368