Reactivity Mapping for ANO2 on Bead-Based Peptide Arrays.

With the use of the PEPscreen Library Design Tool (Sigma-Aldrich), a total of 34 N-terminally biotinylated peptides derived from ANO2 fragment-A (region 79–167) were designed to be 15- or 20-aa long with a 12- or 10-aa overlap, respectively. In accordance with a recently described protocol (38), each biotinylated peptide (50 µM) was coupled on magnetic beads coated with NeutrAvidin (Thermo Scientific) (250 µg/mL). Following the previously described assay steps, including a preadsorption against NeutrAvidin-specific plasma antibodies (38), the bead-based peptide array was used for multiplex mapping of the IgG reactivity against ANO2 in a randomly chosen subset of plasma samples consisting of 185 MS cases and 178 controls.