Nanoimmunosensor based on atomic force spectroscopy to detect anti-myelin basic protein related to early-stage multiple sclerosis.

Multiple Sclerosis (MS) is a chronic inflammatory disorder in the central nervous system for which biomarkers for diagnosis still remain unknown. One potential biomarker is the myelin basic protein. Here, a nanoimmunosensor based on atomic force spectroscopy (AFS) successfully detected autoantibodies against the MBP85-99 peptide from myelin basic protein. The nanoimmunosensor consisted of an atomic force microscope tip functionalization with MBP85-99 peptide, which was made to interact with a mica surface coated either with a layer of anti-MBP85-99 (positive control) or samples of cerebrospinal fluid (CSF) from five multiple sclerosis (MS) patients at different stages of the disease and five non-MS subjects. The adhesion forces obtained from AFS pointed to a high concentration of anti-MBP85-99 for the two patients at early stages of relapsing-remitting multiple sclerosis (RRMS), which were indistinguishable from the positive control. In contrast, considerably lower adhesion forces were measured for all the other eight subjects, including three MS patients with longer history of the disease and under treatment, without episodes of acute MS activity. We have also shown that the average adhesion force between MBP85-99 and anti-MBP85-99 is compatible with the value estimated using steered molecular dynamics. Though further tests will be required with a larger cohort of patients, the present results indicate that the nanoimmunosensor may be a simple tool to detect early-stage MS patients and be useful to understand the molecular mechanisms behind MS.

A highly specific and sensitive nanoimmunosensor for the diagnosis of neuromyelitis optica spectrum disorders.

A precise diagnosis for neuromyelitis optica spectrum disorders (NMOSD) is crucial to improve patients' prognostic, which requires highly specific and sensitive tests. The cell-based assay with a sensitivity of 76% and specificity of 100% is the most recommended test to detect anti-aquaporin-4 antibodies (AQP4-Ab). Here, we tested four AQP4 external loop peptides (AQP461-70, AQP4131-140, AQP4141-150, and AQP4201-210) with an atomic force microscopy nanoimmunosensor to develop a diagnostic assay. We obtained the highest reactivity with AQP461-70-nanoimunosensor. This assay was effective in detecting AQP4-Ab in sera of NMOSD patients with 100% specificity (95% CI 63.06-100), determined by the cut-off adhesion force value of 241.3 pN. NMOSD patients were successfully discriminated from a set of healthy volunteers, patients with multiple sclerosis, and AQP4-Ab-negative patients. AQP461-70 sensitivity was 81.25% (95% CI 56.50-99.43), slightly higher than with the CBA method. The results with the AQP461-70-nanoimmunosensor indicate that the differences between NMOSD seropositive and seronegative phenotypes are related to disease-specific epitopes. The absence of AQP4-Ab in sera of NMOSD AQP4-Ab-negative patients may be interpreted by assuming the existence of another potential AQP4 peptide sequence or non-AQP4 antigens as the antibody target.

Antibody-mediated biorecognition of myelin oligodendrocyte glycoprotein: computational evidence of demyelination-related epitopes.

Antigen-antibody interaction is crucial in autoimmune disease pathogenesis, as multiple sclerosis and neuromyelitis optica. Given that, autoantibodies are essential biomolecules, of which the myelin oligodendrocyte glycoprotein (MOG) can figure as a target. Here we combined Molecular Dynamics (MD), Steered Molecular Dynamics (SMD), and Atomic Force Microscope (AFM) to detail MOG recognition by its specific antibody. The complex model consisted of the MOG external domain interacting with an experimental anti-MOG antibody from the Protein Data Bank (1PKQ). Computational data demonstrated thirteen MOG residues with a robust contribution to the antigen-antibody interaction. Comprising five of the thirteen anchor residues (ASP102, HIS103, SER104, TYR105, and GLN106), the well-known MOG92-106 peptide in complex with the anti-MOG was analysed by AFM and SMD. These analyses evidenced similar force values of 780 pN and 765 pN for computational and experimental MOG92-106 and anti-MOG detachment, respectively. MOG92-106 was responsible for 75% of the total force measured between MOG external domain and anti-MOG, holding the interaction with the antibody. The antigen-antibody binding was confirmed by Surface Plasmon Resonance (SPR) measurements. Combined approaches presented here can conveniently be adjusted to detail novel molecules in diseases research. This can optimize pre-clinical steps, guiding experiments, reducing costs, and animal model usage.