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a Université Paris-Descartes, Faculté de Médecine, UPRES-EA 4058, Paris, France b Service de Médecine Interne, Centre de Référence pour les vascularites nécrosantes et la sclérodermie systémique, Université Paris-Descartes, Faculté de Médecine, Hôpital Cochin, Assistance Publique–Hôpitaux de Paris, Paris, France c Servicio de Reumatologia, Hospital Ramon y Cajal, Madrid, Spain d Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France e Inserm, U567, Paris, France
Key Words: electrophoresis • immunoblotting • proteomics • autoantigen • autoantibody
Address for correspondence: Dr. Luc Mouthon, UPRES-EA 4058, Pavillon Gustave Roussy, 4e étage, Hôpital Cochin, 8 rue Méchain, 75014, Paris, France. Voice: +33-144-41-2544; fax: +33-144-41-2546. luc.mouthon{at}cch.aphp.fr
We here present a method for the identification of antigens recognized by autoantibodies in healthy individuals and patients with autoimmune diseases. We have analyzed IgG reactivities from healthy individuals and patients with autoimmune diseases with endothelial cell antigens by combining a one-dimensional (1D) quantative immunoblotting technique and a 2D immunoblotting technique. Whole-cell protein extracts obtained from human umbilical vein endothelial cells (HUVEC) were used as a source of antigens. Serum IgG from healthy blood donors and from patients with autoimmune diseases were tested at a concentration of 200 µg/mL. One-dimensional immunoblotting was first performed to detect candidate reactivity bands and 2D immunoblotting was secondly performed following 2D electrophoresis to identify protein spots. The gels and 2D blots were scanned and analyzed by imaging software. The matching permitted exact localization of particular relevant protein spots hybridized by antibodies on the 2D blots. The targeted bands from 1D spots and the targeted spots from 2D gels were identified by Edman's N-terminal sequencing and mass spectrometry, respectively. This approach is applicable to both normal and pathological conditions, potentially leading to the identification of new relevant target antigens.
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