We are especially grateful to Dr M. a BIAcore. In this system grouping of 12 of the 13 anti-PR3 MoAbs based on their mutual acknowledgement patterns was achieved. Four MoAbs, from different research groups, namely 12.8, PR3G-2, 6A6 and Hz1F12, recognized comparable epitopes (group 1). Group 2 MoAbs including PR3G-4 and Ganirelix PR3G-6 bound to overlapping regions on PR3. The MoAbs PR3G-3, 4A5 and WGM2 acknowledged similar epitopes Ganirelix as they inhibited binding of each other (group 3). The fourth group of related MoAbs consisted of MC-PR3-2, 4A3 and WGM3. Because of its binding characteristics MoAb WGM1 could not be grouped. These results demonstrate that eight well-established anti-PR3 MoAbs produced by different research groups and four newly produced anti-PR3 MoAbs recognize four individual epitope areas on PR3, including one area detected with newly raised MoAbs only. Keywords: ANCA, proteinase 3, epitope mapping, biosensor INTRODUCTION Autoantibodies directed against cytoplasmic proteins of neutrophilic granulocytes and monocytes (so called anti-neutrophil cytoplasmic autoantibodies (ANCA)) are a highly sensitive and specific marker for Wegener’s granulomatosis (WG) [1,2]. The major antigen recognized by ANCA in WG has been identified as PR3 [3C5], an elastinolytic enzyme originally explained by Baggliolini in 1978 [6]. PR3 is usually a neutral serine proteinase, which is usually localized in the azurophilic granules of neutrophils and in granules of monocytes [7]. data suggest functional activities Ganirelix of the autoantibodies in vivo. Three functional characteristics of ANCA have been implicated in the pathogenesis of WG. First, ANCA are able to activate primed neutrophils to produce oxygen radicals and release lytic enzymes, including PR3 [13C15]. Second, PR3-ANCA can interfere with the binding of PR3 to its physiological inhibitor 1-antitrypsin (1-AT) [16C18]. Third, PR3-ANCA can interfere with the proteolytic activity of PR3 [16,17]. These interfering antibodies may thus act as option inhibitors. However, at the site of inflammation PR3 can cleave the complex between these inhibiting ANCA and PR3 itself, leaving active PR3 [19]. The latter two functional characteristics of ANCA, that is interference of ANCA with the binding of PR3 to 1-AT and interference with the proteolytic activity of PR3, have been shown to correlate with disease activity of WG [17,18]. Changes in these functional characteristics of ANCA have been suggested to follow changes in disease activity more accurately than the previously mentioned changes in ANCA titres alone [18,20,21]. This may indicate that changes in epitope specificity of these ANCA resulting in changes in functionality occur during the course of the disease [22]. Little is known about the epitopes on PR3 recognized by ANCA. Epitope analysis of PR3-ANCA has been hampered by the fact that the majority of PR3-ANCA recognizes conformational epitopes on PR3 [23]. Some groups have tried to elucidate these epitopes recognized by PR3-ANCA by means of overlapping linear peptides of the whole sequence of PR3. Williams et al. [24] recognized numerous antigenic sites on PR3 that were surface-accessible. However, background binding of sera was high and control antibodies also bound some of the peptides. In a comparable test system Chang et al. could not reproduce these results [25]. Griffith et al. found that PR3-ANCA from different patients with active vasculitis bound to linear peptides of PR3 in a highly restricted manner. The bound peptides were surface-accessible and one even coincided with the catalytic site [26]. However, these peptides did not correspond to those recognized by Williams. To further define epitopes recognized by PR3-ANCA well-characterized MoAbs to PR3 can serve as tools, as has been shown for antibodies to myeloperoxidase (MPO), another major ANCA antigen in systemic vasculitis [27]. These well-characterized MoAbs can be used in inhibition studies with a large panel of PR3-ANCA sera in order to assess possible epitope shifts in relation to disease activity. In addition, many established anti-PR3 MoAbs, from different research groups, are used for the detection of PR3-ANCA in capture ELISA systems in clinical practice [28C31]. Establishing the epitopes on PR3 that are recognized by those MoAbs is usually therefore also important in order to exclude possible inaccurate results due to interference of the MoAbs with the binding of patient sera to PR3. The IGLC1 purpose of this study was to determine the epitope areas recognized by a large number of established and newly developed MoAbs to PR3. Specificity of the MoAbs for PR3 was analysed. Epitope restriction of eight established and four newly developed MoAbs to PR3 was determined by biosensor technology. The data offered suggest that those 12 anti-PR3 MoAbs identify a restricted quantity of four epitope areas on PR3, including one area detected with newly raised MoAbs only. MATERIALS AND METHODS MoAbs All commonly used MoAbs to PR3 from different research groups were used. In this study MoAb 12.8 has been produced by Goldschmeding et.