Figure 3

Luminex direct binding assay. Microspheres with covalently attached toxins and irrelevant targets were mixed with purified sdAbs at concentrations of 0, 3.4, 10.9, 30.9, 92.6, 277.8, 833.3, and 2500 nM to test the binding affinity. Microspheres were sorted using the Luminex 100 and the median fluorescence signals were obtained. The ricin binder showed that a maximal binding concentration (Vmax) for ricin and ricin A was around 300 nM with < 0.1% of non-specific binding to ricin B, CT, SEB and BoNT/A complex toxoid (A). BoNT/A complex toxoid binders, P4BF7-3 and P4BH8, exhibited the same Vmax. P4BF7-3 had approximately 13% and 25% of the non-specific binding signal to CT and ricin, respectively (B) and P4BH8 had even higher non-specific binding signals than P4BF7-3 (C). SEB binder P1SD3-3 had Vmax around 10.9 nM with less than 1% of non specific binding signals to CT and ricin (D), while P2SC8 had Vmax around 300 nM with more than 30% of non-specific binding signals (E).