2D immunomic approach for the study of IgG autoantibodies in the experimental model of multiple sclerosis Alessia Farinazzo a,1 , Beatrice Gini a,1 , Alberto Milli b , Francesca Ruffini c , Silvia Marconi a , Ermanna Turano a , Elena Anghileri a , Francesca Barbieri a , Daniela Cecconi b , Roberto Furlan c , Bruno Bonetti a, ⁎ a Department of Neuroscience, University of Verona, Italy b Department of Biotechnology, University of Verona, Italy c Neuroscience, DIBIT, San Raffaele Hospital, Milan, Italy abstract article info Article history: Received 10 July 2010 Received in revised form 14 September 2010 Accepted 4 October 2010 Keywords: Immunomics 2D-PAGE Autoantibodies Molecular mimicry Experimental autoimmune encephalomyelitis ELISA 2D-immunomics may be useful in the identification of autoantigens in neurological autoimmune diseases, but its application may be limited by denaturation of target proteins. Here we compared the capacity of a single or multiple antigens to elicit autoantibodies targeting multiple neural autoantigens by ELISA and 2D- immunomics. We induced experimental autoimmune encephalomyelitis (EAE) with MBP peptide 89–104 , total MBP or spinal cord homogenate. Both techniques showed anti-MBP IgG only after immunization with total MBP. In addition, 2D-immunomics revealed the presence in EAE mice of autoantibodies targeting other neural proteins, some displaying partial sequence homology with MBP. The present finding by 2D- immunomics of multiple neural proteins targeted by autoantibodies generated by a single antigen may help to explain the complex autoimmune response observed in multiple sclerosis. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Multiple sclerosis (MS) is an autoimmune disease where the pathogenic cascade probably includes a process of molecular mimicry leading to the activation of T and B cell clones, some of which cross- react with self antigens (Hohlfeld and Wekerle, 2001; Hueber et al., 2002). A major goal of neuroimmunology is the identification of autoantigens in autoimmune diseases, which may help the identifi- cation of the foreign antigens and the design of vaccines. As far as MS is concerned, extensive efforts have been spent in the last decades, with no conclusive results (Cross and Stark, 2005; Ziemssen and Ziemssen, 2005; Fraussen et al., 2009). In most of these cases, auto- reactivity to pre-selected, recombinant proteins/peptides was assessed by using ELISA or radio-immunoassay techniques; although autoreactivity to a number of neural proteins has been reported, the specificity of autoreactive IgG remains unclear (for review, see Fraussen et al., 2009). Such methodological approaches have been validated in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, which can be induced upon immunization with myelin proteins or peptides (Furlan et al., 2009). We have recently applied a 2D-immunomic approach in MS (Lovato et al., 2008) and Hashimoto's encephalopathy (HE) (Gini et al., 2008), two autoimmune conditions with production of auto- antibodies towards unknown autoantigens. After 2D separation of human neural proteins and transfer to nitrocellulose membranes, sera and cerebro-spinal fluid (CSF) have been applied and the auto- reactive spots identified. This approach led to the identification of few neural antigens recognized by IgG in the CSF of HE patients (Gini et al., 2008), whereas the situation in MS was highly complex, with a wide array of neural proteins recognized by autoantibodies both in serum and in CSF (Lovato et al., 2008). A great advantage of such technique is represented by the opportunity to use the (almost complete) human neural proteome, with proteins present in all their isoforms with post-translational modifications. We have found evidence that these modifications play an important role both in MS and HE, since single isoforms were specifically recognized by autoreactive IgG, while others were targeted also by controls (Lovato et al., 2008). A major limitation of such approach may be represented by the denaturing conditions of the technique with loss of conformational structure; as a conse- quence, IgG directed to conformational epitopes would probably be under-estimated, when tested against denatured proteins. Here we compared the ability of 2D-immunomic and ELISA techniques to detect the presence of autoantibodies in the serum of Journal of Neuroimmunology 232 (2011) 63–67 ⁎ Corresponding author. Section of Neurology, Department of Neurological Sciences and Vision, University of Verona, Policlinico G.B. Rossi, P.le L.A. Scuro 10, 37134 Verona, Italy. Tel.: + 39 045 8124694; fax: + 39 045 8027492. E-mail address: bruno.bonetti@univr.it (B. Bonetti). 1 These authors equally contributed to the work. 0165-5728/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2010.10.004 Contents lists available at ScienceDirect Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim