Abstract
Diagnostics of Multiple Sclerosis (MS) are essentially based on the gold standard magnetic resonance imaging. Few alternative simple assays are available to follow up disease activity. Considering that the disease can remain elusive for years, identification of antibodies fluctuating in biological fluids as relevant biomarkers of immune response is a challenge. In previous studies, we reported that anti-N-glucosylated (N-Glc) peptide antibodies that can be easily detected in Solid-Phase Enzyme-Linked ImmunoSorbent Assays (SP-ELISA) on MS patients’ sera preferentially recognize hyperglucosylated adhesin of non-typeable Haemophilus Influenzae. Since multivalency can be useful for diagnostic purposes to allow an efficient coating in ELISA, we report herein the development of a collection of Multiple N-glucosylated Peptide Epitopes (N-Glc MEPs) to detect anti-N-Glc antibodies in MS. To this aim, a series of N-Glc peptide antigens to be represented in the N-GlcMEPs were tested in competitive ELISA. We confirmed that the epitope recognized by antibodies shall contain at least 5-mer sequences including the fundamental N-Glc moiety. Using a 4-branched dendrimeric lysine scaffold, we selected the N-Glc MEP 24, carrying the minimal epitope Asn(Glc) anchored to a polyethylene glycol-based spacer (PEG) containing a 19-atoms chain, as an efficient multivalent probe to reveal specific and high affinity anti-N-Glc antibodies in MS.
Highlights
Multiple Sclerosis (MS) is the most frequent, chronic, inflammatory, demyelinating, disabling disease of the central nervous system, mainly caused by an autoimmune response to self-antigens in genetically susceptible individuals
We demonstrated that anti-N-glucopyranosyl moiety (Glc) peptide antibodies, detected by a Solid-Phase (SP)-Enzyme-Linked ImmunoSorbent Assays (ELISA) [22,23], preferentially recognize hyperglucosylated adhesin of non-typeable Haemophilus influenzae (NTHi) the
21-mer peptide (TPRVERN(Glc)GHSVFLAPYGWMVK) that was optimized as a type I’ beta-turn structure because of its ability to expose at the best, at position 7, the minimal, but fundamental moiety Asn(N-Glc), in the epitope for autoantibody recognition in the solid-phase conditions of the immunoenzymatic assay [31]. Starting from this assumption, preliminary experiments were performed to select the shortest peptide sequences corresponding to the epitope(s) to be presented in multiple copies in fully characterized multivalent Multiple Epitope Peptides (MEPs) to detect antibodies in MS patients’ sera by SP-ELISA
Summary
Multiple Sclerosis (MS) is the most frequent, chronic, inflammatory, demyelinating, disabling disease of the central nervous system, mainly caused by an autoimmune response to self-antigens in genetically susceptible individuals. MS diagnosis and prognosis are mainly supported by magnetic resonance imaging (MRI) that up to now is considered the gold standard diagnostic technique [1]. To the best of our knowledge, there is still no biological marker relevant for MS diagnosis and for its prognosis [2,3,4]. A cell-based assay to detect antibodies to myelin oligodendrocyte glycoprotein (MOG), one of the candidate protein autoantigens in MS, has been proposed. The real antigen(s) responsible of anti-MOG antibody recognition in the assay remain elusive
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