Endopeptidase-ELISA

The endopeptidase-ELISA was performed according to Jones et al.⁵⁵ using a slightly modified protocol. 3 µg/mL of rSNAP-25H6 or H6trVAMP-2 diluted in 50 mM carbonate-buffer (pH 9.6) were coated on MaxiSorp microtitre plates overnight at 4 °C. On the next day, coating solution was decanted and plates were blocked with 5% skimmed milk powder PBS-T for 90 minutes at room temperature. Subsequently, plates were washed with ddH₂O (4 × ), dried, and toxin diluted in cleavage buffer (see above) was added. Plates were incubated for 18 h at 37 °C with constant back and forth movement in a hybridisation oven (Biometra GmbH, Göttingen, Germany) for substrate cleavage. Plates were then washed with PBS-T (4×) and incubated with 100 µL neoepitope-specific antibodies diluted to 1 µg/mL in 2% skimmed milk powder PBS-T for 90 minutes at room temperature on a multiwell plate shaker (IKA, Staufen, Germany) at 300 rpm for the detection of cleavage products. After incubation with Neo-mAbs, plates were washed again with PBS-T (4×) and incubated with 100 µL HRP-coupled goat anti-mouse IgG H + L (1:2500 dilution, Dianova, Hamburg, Germany) secondary antibody diluted in 2% skimmed milk powder PBS-T for 30 minutes at room temperature on a multiwell plate shaker at 300 rpm. Finally, plates were washed with PBS-T (8×) and developed as described above.