IgG derived

from a SS patient positive for antibodies to the see more third extracellular loop had no effect on (Ca2+)I, as well as IgG derived from an anti-M3R antibody-negative SS patient (Figs 3e and 4). Recently, anti-M3R antibodies have been the focus of interest in rheumatology because of their potential pathogenic role, use as diagnostic markers and being therapeutic targets in patients with SS [1]. Several methods have been used to detect anti-M3R antibodies in SS patients [1]. In functional assays using smooth muscles, IgG fractions from patients with SS (SS-IgG) inhibited carbachol-evoked or nerve-evoked bladder or colon contractions [8,9]. In salivary gland cells, SS-IgG inhibited the rise in (Ca2+)i induced by carbachol, and also inhibited pilocarpine-induced AQP5 trafficking to the apical membrane from the cytoplasm [2]. The inhibitory actions of SS-IgG on

the rise in (Ca2+)i was acutely reversible [10]. Anti-M3R antibodies from SS patients can be detected by immunofluorescent analysis using rat lacrimal glands [11], and by flow cytometry using the M3R-transfected Chinese hamster ovary (CHO) cell line [12]. Moreover, anti-M3R antibodies in sera of SS patients were detected by ELISA using synthetic peptides or recombinant proteins of the second extracellular loop of M3R [13]. We have reported previously the presence of anti-M3R antibodies in a group of patients with SS, which recognized the second extracellular loop by ELISA using synthetic buy Torin 1 peptides [4,5]. In the present study, we established a standard method to detect anti-M3R antibodies that can be used for screening large patient populations. Functional assays and flow cytometry are too laborious for routine use. Although ELISA is easy, the results from some ELISA systems used for screening anti-M3R antibodies differ Mannose-binding protein-associated serine protease widely with regard to the prevalence of anti-M3R antibodies (from 11 to 90%) [4,14]. Furthermore, Cavill et al.[15]

reported failure to detect anti-M3R antibodies by ELISA using synthetic peptides. In the present study, we reported higher frequencies and titres of anti-M3R antibodies against all extracellular domains in SS patients than the control. The prevalence of anti-M3R antibodies against the second extracellular loop in SS (55%) determined in the present study was much higher than that reported in our previous study (11%) [4]. The reason for this difference is probably related to the change in the methodology, such as increased sensitivity resulting from purity of the synthetic peptides, modification of the washing procedure or other factors introduced in the modified ELISA system. In the present study, we also determined the precise B cell epitopes of M3R molecules.