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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Antigenic Probes for Autoantibody Detection in MultipleSclerosis: Synthetic Peptides versus Recombinant ProteinsFrancesca Gori 1,2 , Barbara Mulinacci 1,2, * , Lara Massai 1,2 ,Francesco Lolli 1,3 , Anna Maria Papini 1,4,5 , and Paolo Rovero 1,21 Laboratory of Peptide & Protein Chemistry & Biology, University of Florence, SestoFiorentino (Fi), I-50019, Italy; 2 Department of Pharmaceutical Sciences, University ofFlorence, Sesto Fiorentino (Fi), I-50019, Italy; 3 Department of Neurological Sciences,University of Florence, Azienda Ospedaliero-Universitaria Careggi, Firenze, I-50134,Italy; 4 Department of Chemistry ‘‘Ugo Schiff’’ and CNR-ICCOM, University of Florence,Sesto Fiorentino (Fi), I-50019, Italy; 5 Laboratoire SOSCO-EA4505, University ofCergy-Pontoise, Cergy-Pontoise cedex, 95031, France* present address: Max-Planck Institut für Biochemie, Martinsried, München, GermanyIntroductionAutoimmune diseases are a class of disorders that need early diagnosis and efficientprognosis for setting up therapeutic treatments. Multiple sclerosis is the most commoncentral nervous system inflammatory demyelinating disease. Its pathogenesis has not beenyet elucidated, but an autoimmune mechanism against myelin antigens is thought tocontribute to its immunopathological mechanisms.The identification of autoantibodies as specific biomarkers is a relevant target and, upto now, most of the diagnostic immunoassays are based on native antigens as immunologicalprobes.One of the most studied antigen targets in Multiple Sclerosis is MyelinOligodendrocyte Glycoprotein (MOG), a glycoprotein of the myelin sheath [1]. MOG isconsidered a putative autoantigen and interesting data are focused on the diagnostic andprognostic role of the detection of antibodies to MOG in adults’ serum [2].However, post-translational modifications of proteins, either native or aberrant, mayplay a fundamental role for specific and sensitive autoantibody detection in autoimmunediseases. So antigens characterization should take into account the chemical modificationintroduced on side chains of amino acids. The use of chemically modified syntheticpeptides can be useful for this purpose.We demonstrated that a ‘chemical reverse approach’ is efficient in developingspecifically modified synthetic peptides, able to fishing out autoantibodies from patients’biological fluids. In particular a specific antigenic probe, termed CSF114(Glc), wasdeveloped to identify autoantibodies, as biomarkers correlating with disease activity, in apopulation of Multiple Sclerosis patients [3,4].We decided to focus our attention on recombinant MOG with the aim of comparingthe relative merit of this protein versus synthetic modified peptides as antigenic probes forautoantibody detection in ELISA.Results and DiscussionThe full-length MOG consists of 218 amino acids; the encephalitogenic properties of thisprotein are believed to result from the extracellular IgV-like domain (amino acids 1-125).The cDNA of the extracellular domain of rat MOG was subcloned into the His-tagexpression vector pQE12. rMOG ED (His) 6 was overexpressed in inclusion bodies in E. coli.After disruption of the cells by sonication, the inclusion bodies were purified by repetitivesteps of centrifugation and resuspension in 50 mM Tris, 0.5 M NaCl, 0.5% lauryldimethylamineoxide, pH 8.0. The inclusion bodies were solubilized in a denaturating buffer (100mM NaH 2 PO 4 , 10 mM Tris, 6 M guanidine HCl, 40 mM mercaptoethanol, pH 8.0).rMOG ED (His) 6 was loaded onto an affinity chromatography column (ChelatingSepharose Fast Flow; GE Healthcare) and the refolding of the protein was achieved under agradient of denaturating buffer (100 mM NaH 2 PO 4 , 10 mM Tris, 6 M guanidine HCl, pH8.0) versus nondenaturating solution (100 mM NaH 2 PO 4 , 10 mM Tris, 3 mM reducedglutathione, pH 8.0) over 10 h. The folded protein was subsequently eluted with 0.5 Mimidazole in 100 mM NaH 2 PO 4 , 10 mM Tris, 0.2 M NaCl (pH 8.0). Finally the protein wasdialyzed against PBS, pH 8.500