Label-free method for anti-glucopeptide antibody detection in Multiple Sclerosis

Surface plasmon resonance technique is particularly interesting in immunology because it has the potential to visualize label-free antigen-antibody interactions in real-time, thus enabling antibody detection and monitoring. Herein we release the guidelines for the correct use of a method to detect specific antibodies directly in Multiple Sclerosis patients’ sera using a glucopeptide-based label-free biosensor. The protocol describes the strategy employed for the immobilization of glucopeptide antigen onto a gold sensor chip and the evaluation of the specific binding of serum antibodies to the immobilized antigen. • Label-free method for the real time screening of disease-specific antibodies within a few minutes; • The described protocol employs small quantities of glucopeptide antigen and blood serum samples saving method-cost; • Stability of the immobilized glucopeptide antigen guarantees the regeneration of the surface allowing re-use the immunosensor with high automated throughput. The antibodies detected using the described methodology can be evaluated as biomarkers of Multiple Sclerosis. The SPR detection system is able to characterize antibodies significantly different from those evaluated in the classical enzyme-linked immunosorbent assays (ELISA)

An Optimised Di-Boronate-ChemMatrix Affinity Chromatography to Trap Deoxyfructosylated Peptides as Biomarkers of Glycation

We report herein a novel ChemMatrix® Rink resin functionalised with two phenylboronate (PhB) moieties linked on the N-α and N-ε amino functions of a lysine residue to specifically capture deoxyfructosylated peptides, compared to differently glycosylated peptides in complex mixtures. The new PhB-Lys(PhB)-ChemMatrix® Rink resin allows for exploitation of the previously demonstrated ability of cis diols to form phenylboronic esters. The optimised capturing and cleavage procedure from the novel functionalised resin showed that only the peptides containing deoxyfructosyl-lysine moieties can be efficiently and specifically detected by HR-MS and MS/MS experiments. We also investigated the high-selective affinity to deoxyfructosylated peptides in an ad hoc mixture containing unique synthetic non-modified peptides and in the hydrolysates of human and bovine serum albumin as complex peptide mixtures. We demonstrated that the deoxyfructopyranosyl moiety on lysine residues is crucial in the capturing reaction. Therefore, the novel specifically-designed PhB-Lys(PhB)-ChemMatrix® Rink resin, which has the highest affinity to deoxyfructosylated peptides, is a candidate to quantitatively separate early glycation peptides from complex mixtures to investigate their role in diabetes complications in the clinics

Erratum: Antibodies from multiple sclerosis patients preferentially recognize hyperglucosylated adhesin of non-typeable Haemophilus influenzae

Scientific Reports 6: Article number: 39430; published online: 23 December 2016; updated: 22 March 2017. The original version of this Article contained typographical errors in the Abstract. ‘In autoimmune diseases, there have been proposals that exogenous “molecular triggers”, i.e., specific this should be ‘non-self antigens’ accompanying infectious agents, might disrupt control of the adaptive immune system resulting in serious pathologies’.</jats:p

Antibodies from multiple sclerosis patients preferentially recognize hyperglucosylated adhesin of non-typeable Haemophilus influenzae

In autoimmune diseases, there have been proposals that exogenous "molecular triggers", i.e., specific this should be 'non-self antigens' accompanying infectious agents, might disrupt control of the adaptive immune system resulting in serious pathologies. The etiology of the multiple sclerosis (MS) remains unclear. However, epidemiologic data suggest that exposure to infectious agents may be associated with increased MS risk and progression may be linked to exogenous, bacterially-derived, antigenic molecules, mimicking mammalian cell surface glycoconjugates triggering autoimmune responses. Previously, antibodies specific to a gluco-asparagine (N-Glc) glycopeptide, CSF114(N-Glc), were identified in sera of an MS patient subpopulation. Since the human glycoproteome repertoire lacks this uniquely modified amino acid, we turned our attention to bacteria, i.e., Haemophilus influenzae, expressing cell-surface adhesins including N-Glc, to establish a connection between H. influenzae infection and MS. We exploited the biosynthetic machinery from the opportunistic pathogen H. influenzae (and the homologous enzymes from A. pleuropneumoniae) to produce a unique set of defined glucosylated adhesin proteins. Interestingly we revealed that a hyperglucosylated protein domain, based on the cell-surface adhesin HMW1A, is preferentially recognized by antibodies from sera of an MS patient subpopulation. In conclusion the hyperglucosylated adhesin is the first example of an N-glucosylated native antigen that can be considered a relevant candidate for triggering pathogenic antibodies in MS