Novel candidate markers for multiple sclerosis using phage cDNA display.

Abstract

Multiple sclerosis (MS) is a chronic, inflammatory disease of the central nervous system, characterized by the presence of focal lesions resulting from myelin breakdown. In the past few years, an important contribution of B cells and autoreactive antibodies has been demonstrated in the pathogenesis of MS. To fully explore the complex information present within the antibody repertoire of patients, we have developed a novel and powerful molecular approach 'Serological Antigen Selection', which involves the display of a cDNA expression library on filamentous phage and subsequent selection on patient IgG. The aim of this study was to apply the SAS technology to identify antigens that are specially recognized by antibodies (IgG) present in the cerebrospinal fluid (CSF) of MS patients. First, we constructed a cDNA display library by cloning a normalized cDNA library prepared for active chronic MS plagues, with varying degrees of demyelinaton and inflammatory activity (Soares et al, 1994) for expression as a fusion protein with a filamentous phage minor coat protein, pVI. Parallel selections were then performed on 2 pools of CSF (n = 10) from relapsing-remitting MS patients. Affinity selections revealed a panel of 9 different clones reactive with the first CSF pool. A detailed serological analysis of the 9 different antigens on a large panel (n = 100) of individual patient and control CSF showed exclusive reactivity to MS patient CSF for seven different antigens. Sequence analysis revealed that these clones have never been associated with MS. Antigenic cDNAs from the second pool of CSF are currently under investigaton. In conclusion, our findings demonstrate that this novel molecular approach is useful to identify novel candidate antigens in MS that can be used as diagnostic markers, and can be used to study the humoral immune response in MS.