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).
Abstract The design of molecules that mimic biologically relevant glycans is a significant goal for understanding important biological processes and may lead to new therapeutic and diagnostic agents. In this study we focused our attention on the trisaccharide human natural killer cell‐1 (HNK‐1), considered the antigenic determinant of myelin‐associated glycoprotein and the target of clinically relevant auto‐antibodies in autoimmune neurological disorders such as IgM monoclonal gammopathy and demyelinating polyneuropathy. We describe a structure–activity relationship study based on surface plasmon resonance binding affinities aimed at the optimization of a peptide that mimics the HNK‐1 minimal epitope. We developed a cyclic heptapeptide that shows an affinity of 1.09×10 −7 m for a commercial anti‐HNK1 mouse monoclonal antibody. Detailed conformational analysis gave possible explanations for the good affinity displayed by this novel analogue, which was subsequently used as an immunological probe. However, preliminary screening indicates that patients′ sera do not specifically recognize this peptide, showing that murine monoclonal antibodies cannot be used as a guide to select immunological probes for the detection of clinically relevant human auto‐antibodies.
Antibody detection in autoimmune disorders, such as multiple sclerosis (MS) and Rett syndrome (RTT) can be achieved more efficiently using synthetic peptides. The previously developed synthetic antigenic probe CSF114(Glc), a type I' β-turn N-glucosylated peptide structure, is able to recognize antibodies in MS and RTT patients' sera as a sign of immune system derangement. We report herein the design, synthesis, conformational analysis, and immunological evaluation of a collection of glycopeptide analogs of CSF114(Glc) to characterize the specific role of secondary structures in MS and RTT antibody recognition. Therefore, we synthesized a series of linear and cyclic short glucosylated sequences, mimicking different β-turn conformations, which were evaluated in inhibition enzyme-linked immunosorbent assays (ELISA). Calculated IC50 ranking analysis allowed the selection of the candidate octapeptide containing two (S)-2-amino-4-pentynoic acid (L-Pra) residues Ac-Pra-RRN(Glc)GHT-Pra-NH2 , with an IC50 in the nanomolar range. This peptide was adequately modified for solid-phase ELISA (SP-ELISA) and surface plasmon resonance (SPR) experiments. Pra-RRN(Glc)GHT-Pra-NH2 peptide was modified with an alkyl chain linked to the N-terminus, favoring immobilization on solid phase in SP-ELISA and differentiating IgG antibody recognition between patients and healthy blood donors with a high specificity. However, this peptide displayed a loss in IgM specificity and sensitivity. Moreover, an analog was obtained after modification of the octapeptide candidate Ac-Pra-RRN(Glc)GHT-Pra-NH2 to favor immobilization on SPR sensor chips. SPR technology allowed us to determine its affinity (KD = 16.4 nM), 2.3 times lower than the affinity of the original glucopeptide CSF114(Glc) (KD = 7.1 nM).
Abstract The present work is a preliminary study on the potential of low-frequency ultrasound irradiation coupled with O 3 process for the disinfection of a primary effluent from a municipal wastewater treatment plant preserving nutrient levels (in particular nitrogen and phosphorous), for its possible reuse in civil, industrial, and agricultural sectors. The treated water could be reused, after appropriate dilution, contributing to the circular economy perspective and reducing the need for both chemical fertilizer addition and freshwater supply. The effect of different specific ultrasonic energies and ozone doses was assessed on a bench-top system, composed of an ultrasonic reactor and a semi-batch ozonation vessel. The results showed that the combined US-O 3 process produces a good removal efficiency regarding soluble Chemical Oxygen Demand, sCOD (ca. 60%), anionic surfactants (ca. 50%), and formaldehyde (ca. 50%), and an optimal abatement for Methylene Blue Active Substances (MBAS, > 90%). The process also reached high disinfection performances, obtaining 4 logs for E. coli and 5 log abatement for Total Coliforms. The high removal efficiency is matched by an outstanding retention of nutrients (total nitrogen and orthophosphate) highlighting a high potential value for agricultural reuse of the treated primary effluent, with possible significant saving of chemical fertilizers. It was concluded that low-frequency ultrasound pre-treatment, combined with ozonation, could be a useful process for primary effluent recovery for several purposes. Further studies are expected to be planned and executed to evaluate system scale-up feasibility.
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. 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.
