Fig. 1

Primary and secondary enzyme screening assays. Primary 96-well plate assay (10 μM S-(−)-nicotine): (a) The activity of a fixed amount of NicA2-His (green) captured by anti-His mAb (red) immobilized in each assay well is monitored by the Amplex Red assay measuring the formation of the fluorescent product Resorufin over time, (b) An activity assay using purified proteins comparing wt NicA2 (open circle) to NicA2Δ50 (open square) and NicA2Δ50W427Q (solid diamond). The relative slopes of the curves of Relative Fluorescence Units (RFU) vs. time were 1, 0.7 and 3.6, respectively. Secondary biosensor assay (250 nM S-(−)-nicotine): (c) Sensorgram of 3-fold serial dilution of S-(−)-nicotine in running buffer passed over a sensor surface with immobilized anti-nicotine antibody over a range of nicotine concentrations (as indicated, in triplicate). After a steady-state level of binding was achieved, running buffer without nicotine was passed over the chip (starting at t = 240 s) to dissociate nicotine from the mAb. (d) Plot of sensor Response Units (RU) at steady-state (from experiment in panel C) vs. S-(−)-nicotine concentration