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Sophisticated approaches have recently led to the identification of novel autoantigens associated with Multiple Sclerosis (MuS), e.g. neurofascin, contactin, CNPase, and other T-cell receptor membrane anchored proteins. These putative antigens, although differing from the conventional myelin derivatives, are conceptually based on an animal model of experimental autoimmune encephalomyelitis. In this report we describe the identification of putative antigens based on their recognition by autoantibodies isolated from MuS patient serum. In a previous work from this laboratory we have shown that a peptide probe, named CSF114(Glc), specifically identifies serum autoantibodies in a subset of MuS patients, representing ∼30% of the patient population. The autoantibodies, purified from MuS patients' sera (six), through CSF114(Glc) affinity chromatography, detected three immunoreactive protein bands present in the rat brain. Proteomic analysis of the immunoreactive bands, involving MALDI and MS/MS techniques, revealed the presence of four proteins distinguishable by their mass: alpha fodrin, alpha actinin 1, creatine kinase, and CNPase. The immunoreactive profile of these rat brain proteins was compared with that of commercially available standard proteins by challenging against either CSF114(Glc) purified MuS autoantibodies, or monoclonal antibodies. Further discrimination among the rat brain proteins was provided by the following procedure: whereas monoclonal antibodies recognized all rat brain proteins, isolated MuS specific antibodies recognize only alpha actinin 1 as a putative antigen. In fact, alpha actinin 1 displayed a robust immunoreactive response against all MuS patients' sera examined, whereas the other three bands were not consistently detectable. Thus, alpha actinin 1, a cytoskeleton protein implicated in inflammatory/degenerative autoimmune diseases (lupus nephritis and autoimmune hepatitis) might be regarded as a novel MuS autoantigen, perhaps a prototypic biomarker for the inflammatory/degenerative process typical of the disease.

We previously reported that CSF114(Glc) detects diagnostic autoantibodies in multiple sclerosis sera. We report herein a bioinformatic analysis of myelin proteins and CSF114(Glc), which led to the identification of five sequences. These glucopeptides were synthesized and tested in enzymatic assays, showing a common minimal epitope. Starting from that, we designed an optimized sequence, SP077, showing a higher homology with both CSF114(Glc) and the five sequences selected using the bioinformatic approach. SP077 was synthesized and tested on 50 multiple sclerosis patients' sera, and was able to detect higher antibody titers as compared to CSF114(Glc). Finally, the conformational properties of SP077 were studied by NMR spectroscopy and structure calculations. Thus, the immunological activity of SP077 in the recognition of specific autoantibodies in multiple sclerosis patients' sera may be ascribed to both the optimized design of its epitopic region and the superior surface interacting properties of its C-terminal region.

In multiple sclerosis (MS) the gold standard for the diagnosis and prognosis is, up to now, the use of magnetic resonance imaging markers. No alternative simpler assays proven of use, except for cerebrospinal fluid analysis, have been provided in MS diagnosis. Therefore, there is a need to develop non-invasive, sensitive, simple new techniques for the clinical routine. Herein we present the evaluation of the feasibility of a glycopeptide-based biosensor to detect MS specific antibodies in sera using the surface plasmon resonance technology. The previously described glycopeptide antigen CSF114(Glc) has been immobilized on a gold sensor chip and the method has been optimized for real-time specific autoantibody detection directly in sera. A population of 60 healthy blood donors and 61 multiple sclerosis patients has been screened. The receiver operating characteristic (ROC)-based analysis has established the optimal diagnostic cut-off value for the method obtaining a sensitivity of 36% and a specificity of 95%. Sample sera have been also screened with a previously validated ELISA.

Antibodies to MOG in serum have a dubious prognostic value in multiple sclerosis. The MOG recombinant protein conformational properties relevant to the antigenic activity are unknown. We employed a solid-phase ELISA based on a product (rMOG(ED)(His)(6)) expressed in E. coli after subcloning the cDNA of the extracellular domain of rat MOG, performing a refolding procedure on column and affinity purification. The far-UV Circular Dichroism (CD) spectra of rMOG(ED)(His)(6) showed a β-sheet, a characteristic feature of the Ig-fold. However, in MS sera and controls we failed to detected IgM or IgG antibodies., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We report the design, synthesis, and immunological evaluation of a series of glycopeptide analogues of the previously described antigenic probe CSF114(Glc), with the aim of understanding the importance of N-glycosylation on Asn residue in multiple sclerosis antibody recognition. The glucopeptide, characterized by a β-turn conformation which is fundamental for a correct presentation of the epitope, has been modified by introducing various natural glycoamino acids in position 7. The new glycopeptides were evaluated by measuring the IgG and IgM antibody titer in multiple sclerosis patients' and normal blood donors' sera. Moreover, we achieved the efficient synthetic strategy of new Asn derivative bearing N-acetylneuraminic acid (Neu5Ac), linked by an N-glycosidic bond, on the side chain of the Asn residue orthogonally protected for Fmoc/tBu SPPS., (2010 Wiley Periodicals, Inc.)

Circulating autoantibodies have been recognized as disease biomarkers of autoimmune diseases. We have previously disclosed a synthetic glycopeptide that is able to detect specific autoantibodies in sera of patients who are affected by multiple sclerosis (MS). This glycopeptide is characterized by a type I' beta-turn around the minimal epitope Asn(Glc) that allows an efficient exposure of this moiety to antibody interactions in the context of a solid-phase immunoenzymatic assay. With the aim of optimizing the glycopeptide-antibody interactions, we analyze a series of new glycopeptides based on different turn structures. Our results confirm the role of conformation in the recognition and binding of synthetic antigenic probes to MS autoantibodies. Glycopeptide 2, which is characterized by a type I beta-turn around the minimal epitope Asn(Glc), shows the highest antibody affinity (IC50 = 11.8 nM), and thus it appears to be a promising tool for the detection of specific autoantibodies as MS biomarker in patients' sera.

Glycopeptide analogues of CSF114(Glc), modified at N-terminus with new ferrocenyl carboxylic acid and a new ferrocenyl-thiphosphino amino acid, were used to implement a new electrochemical biosensor for autoantibody detection in multiple sclerosis. The ferrocenyl moiety of these "electrochemical probes" did not affect autoantibody recognition both in SP-ELISA and in inhibition experiments. By electrochemical monitoring the interactions of the modified peptides Fc-CSF114(Glc) and 4-FcPhP(S)Abu-CSF114(Glc) with the autoantibodies, we demonstrated that autoantibodies could be detected with a sensitivity comparable to ELISA method. The new electrochemical probes can be proposed to characterize autoantibodies as biomarkers of multiple sclerosis by a simple, rapid, and reproducible cyclic voltammetry-based diagnostic methodology., (Copyright (c) 2008 Wiley Periodicals, Inc.)

Synthetic glycopeptides have the potential to detect antibodies in multiple sclerosis (MS). In the present study, we analyzed the antibodies (IgM class, IgG class and IgG subclasses) to the synthetic glycopeptide CSF114(Glc) in the serum of 186 MS patients, 166 blood donors (BDs), 25 patients affected by meningitis/encephalitis, 41 affected by systemic lupus erythematosus (SLE) and 49 affected by rheumatoid arthritis (RA). The IgM antibody level to CSF114(Glc) was significantly increased in MS patients versus BDs (p<0.001) or versus other autoimmune diseases (SLE or RA, p<0.001). The IgG response was restricted to the subclass IgG2. IgM antibodies to CSF114(Glc) were found in 30% of relapsing/remitting MS patients and, at lower levels, in subjects affected by meningitis/encephalitis. The study of antibodies to CSF114(Glc) is a new, potential immunological marker of MS.

Multiple sclerosis (MS) is a complex disease that seems to depend on several pathophysiological processes. Because of its varied clinical presentation, natural history, and response to therapeutic interventions, MS can be considered to be a group of diseases that have not been yet characterized, thus resulting in difficult evaluation of prognosis. In the last few years, the role of autoAbs in MS has been reevaluated, and, therefore, their identification as specific biomarkers became a relevant target. In this paper, we demonstrate that an aberrant N-glucosylation is a fundamental determinant of autoAb recognition in MS. Thus, we developed CSF114(Glc), an antigenic probe accurately measuring IgM autoAbs in the sera of a patient population, as disease biomarker. The relevance of CSF114(Glc) is demonstrated by its clinical application and correlation with disease activity and prognosis. In fact, CSF114(Glc), a structure-based designed glycopeptide, is able to recognize, by ELISA, the presence of specific IgM autoAbs in the sera of a MS patient population but not in blood donors and other autoimmune conditions. AutoAbs specific for CSF114(Glc) isolated from MS patients recognized myelin and oligodendrocyte antigens by immunohistochemistry but not other nonrelevant tissues. We demonstrate that CSF114(Glc) is a reliable, specific probe in a longitudinal study of untreated MS patients. Development of IgG/IgM anti-CSF114(Glc) Abs paralleled clinical activity and brain lesions positive to MRI. Therefore, a CSF114(Glc)-based immunoassay on sera may have important prognostic value in monitoring MS disease progression guiding optimal therapeutic treatment.