An important contribution of B cells and autoantibodies has been demonstrated in the pathogenesis of multiple sclerosis (MS), leading to interest in the use of such autoantibodies as diagnostic or prognostic biomarkers. The objective of this study was to identify novel Ab biomarkers for MS using "serological Ag selection". Using a phage display library derived from MS brain plaques, we applied serological Ag selection to identify antigenic targets specifically interacting with Abs present in the cerebrospinal fluid (CSF) of 10 relapsing-remitting MS patients. These antigenic targets were further evaluated on a large panel of CSF from 63 other MS patients, 30 patients with other inflammatory disorders, and 64 patients with noninflammatory neurological disorders. A panel of eight antigenic targets was identified that showed a 86% specificity and 45% sensitivity in discriminating MS patients and controls. Four of the antigenic targets showed exclusive reactivity (100% specificity; 23% sensitivity) in the MS group as compared with the control group. Detailed bio-informatic analyses revealed a novel Ag, SPAG16. Among the novel phage peptides identified, novel epitopes were generated from untranslated sequences and out-of-frame sequences. Of 10 relapsing-remitting patients used for serological Ag selection, Ab reactivity toward one of the eight antigenic targets was also demonstrated in serum of 38% CSF-positive patients. Autoantibody profiles against epitopes derived from MS brain tissue could serve as diagnostic markers or form the basis for the identification of a subgroup of MS patients.