mAb inhibition of BoNT/A LC endopeptidase activity.

<p><b>A</b>. SDS-PAGE-based substrate cleavage assay: BoNT/A LC (25 nM) and 20 molar excess of mAb were mixed in Tris buffer (50 mM, pH 8.0). GST-SNAP-25 (141–206) peptide substrate (5 μM) was added to initiate the reaction. Images of Coomassie-stained SDS-PAGE gels of the results after 5 min (the upper panel) or 15 min (lower panel) incubation with the intact substrate and cleaved product indicated by arrows. Ability to inhibit SNAP-25 cleavage was scored as positive (+) or negative (-). Additional bands in the SDS-PAGE gel likely represent GST-SNAP25 breakdown products or impurities from the mAbs. <b>B.</b> Plot of the results from the FRET screening assay for inhibition of substrate cleavage. The YsCsY substrate (2 μM) was mixed with each of the indicated mAbs (200 nM) and BoNT/A LC. (400 pM). The mean (± standard deviation) of the ratio of emissions at 527 nm to 480 nm after 5 min or 15 min are shown. Ratios >0.8 at 15 minutes were interpreted to indicate inhibition of BoNT/A-mediated cleavage by the mAb, denoted by (+). The epitope clusters (I-IV, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135306#pone.0135306.g002" target="_blank">Fig 2</a>) are shown below each scFv. Nine mAbs in epitope cluster I (1C10, 1D8 1G11, 1H5, 10B12, 10C9, 10F9, 10H10 and 11D8) strongly inhibited BoNT/A LC cleavage.