Research paper Detection of one V H antibody sequence in both healthy donors and urticaria patients Michaela Fux a , Monique Vogel a, * , Michael B. Stadler a , Beda M. Stadler a , Sylvia M. Miescher a,b a Institute of Immunology, University of Bern, Inselspital, Sahli-Haus 2, CH-3010 Bern, Switzerland b ZLB Behring AG, Wankdorfstrasse 10, CH-3022 Bern, Switzerland Received 16 February 2005; received in revised form 6 July 2005; accepted 22 September 2005 Available online 25 October 2005 Abstract We have previously isolated anti-FcERIa autoantibodies from phage libraries of healthy donors and urticaria patients. Strikingly, the same antibody, LTMa15, was isolated from both libraries. Sequence analysis revealed a germline configuration of the LTMa15 variable heavy (V H ) chain with a slightly mutated variable light (V L ) chain supporting its classification as a natural autoantibody. Distribution analysis of anti-FcERIa autoantibodies by functional or serological tests delivered conflicting data. For this reason we have developed a new real-time PCR to analyse the distribution of LTMa15V H in healthy donors and urticaria patients. Our new bioinformatic program permitted the design of a minor groove binder (MGB) Taq Man probe that specifically detected the LTMa15V H . We were able to demonstrate a broad range of rearranged V H gene copy number without any correlation to the state of health. Monitoring LTMa15V H gene copy number in a single donor over a period of 70 days revealed a time-related fluctuation of circulating B cells carrying LTMa15V H . We propose that our real-time PCR may serve as a model for the quantification of natural antibody sequences at a monoclonal level. D 2005 Elsevier B.V. All rights reserved. Keywords: Natural autoantibody; Germline configuration; Bioinformatics; Real-time PCR; Minor groove binder TaqMan probe 1. Introduction Chronic idiopathic urticaria (CIU) is defined by the occurrence of daily wheals and itching for at least 6 weeks. The wheals, redness and itching of CIU are due to the release of histamine and other mediators from dermal mast cells. Evidence of the involvement of functional autoantibodies against the a-chain of the high-affinity IgE receptor (FcERIa) of dermal mast cells and basophils in CIU was first reported by Hide et al. (1993, 1994). Such anti-FcERIa autoantibodies were suggested to be a selective marker for a subset of urticaria patients (Fiebiger et al., 1995). However, we have previously reported the presence of anti-FcERIa autoantibodies in the serum of healthy donors (Pachlop- nik et al., 2004) as well as in multidonor intravenous IgG (IVIg) preparations (Horn et al., 1999). Further- more we have isolated anti-FcERIa autoantibodies (LTMa15, UMa16, UGa8) using phage display tech- 0022-1759/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2005.09.014 Abbreviations: V H , variable heavy chain; V L , variable light chain; CDR, complementary determining region; CIU, chronic idiopathic urticaria; FcERIa, a-chain of the high-affinity IgE receptor; C T , threshold cycle number; MGB, Minor groove binder; PBGD, porpho- bilinogen deaminase. * Corresponding author. Tel.: +41 31 632 03 34; fax: +41 31 381 57 35. E-mail address: monique.vogel@iib.unibe.ch (M. Vogel). Journal of Immunological Methods 307 (2005) 107 – 117 www.elsevier.com/locate/jim