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A Combined SDS‐PAGE and Proteomics Approach to Identify Target Autoantigens in Healthy Individuals and Patients with Autoimmune Diseases
Author(s) -
GUILPAIN PHILIPPE,
SERVETTAZ AMÉLIE,
TAMBY MATHIEU C.,
CHANSEAUD YOURI,
TAMAS NICOLAS,
PENALEFEBVRE PALOMA GARCIA de la,
BROUSSARD CÉDRIC,
GUILLEVIN LOÏC,
CAMOIN LUC,
MOUTHON LUC
Publication year - 2007
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1196/annals.1398.060
Subject(s) - autoantibody , antigen , blot , proteomics , microbiology and biotechnology , antibody , autoimmune disease , two dimensional gel electrophoresis , gel electrophoresis , protein microarray , biology , subclass , immunology , chemistry , microarray , biochemistry , gene , gene expression
Abstract : 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.