Inhibitory Effects of Peptides on Stx2a Binding

The inhibitory effects of a series of shorter MMβA-mono-derived peptides on binding between the Stx2a A-subunit and MMβA-mono were measured using the AlphaScreen assay, as described previously^{18 (link)}. Briefly, biotinylated MMβA-mono (30 nM) was incubated with Stx2a A-subunit (20 nM) in the presence of indicated concentrations of a single shorter peptide and specific anti-Stx2a A-subunit monoclonal antibody (originally obtained) in individual wells of an OptiPlate-384 (PerkinElmer, Waltham, MA, USA) for 30 min at room temperature. Samples were then incubated with anti-IgG (protein A) acceptor beads (20 µg/ml; PerkinElmer) for 30 min, followed by incubation with streptavidin donor beads (20 µg/ml; PerkinElmer) for 1 h at room temperature in the dark. The plate was then subjected to excitation at 680 nm, and emission from wells was monitored at 615 nm with the EnVision system (Perkin Elmer). Data were obtained as arbitrary units (AUs) of signal intensity (counts per second). IC50 values were determined by using Image J software ver. 1.53 k.