Fig 1: In vitro assays that will be used to measure venom activity and neutralisation by ADDomers. (a) The SVMP assay uses a quenched fluorogenic peptide substrate (ES010, BioTechne) which is cleaved by SVMPs, resulting in a free unquenched fragment. Structural model shown (green-pink-cyan) is an E. ocellatus SVMP (AlphaFold AF-Q2UXQ5-F1). (b) Enzymatically active PLA2s are measured using a fluorescent assay (EnzCheck, ThermoFisher) in which a fluorescent substrate (BODIPY PC-A2) is encapsulated in liposomes. PLA2s hydrolyse the phospholipids, resulting in increased fluorescence. Structural model shown (blue) is a PLA2 from E. ocellatus (AlphaFold AF-P59171-F1). (c) The plasma clotting assay measures the optical density (OD) of plasma in a spectrophotometer to detect clot formation where a higher OD indicates clotting. A normal plasma clotting control is concurrently run, and compared to venom samples to indicate whether venom is causing pro-coagulant or anti-coagulant effects. (d) Flow cytometry assays can demonstrate disturbances in platelet aggregation (indicated by changes to forward and side scatter) and the binding of disintegrins to activated GPIIb/IIIa can be detected with PAC-1-FITC antibody. (e) The neurotoxic activity of α-neurotoxins (a subclass of 3FTx) can be measured in a cell-based assay using cells natively expressing muscle-type nAChRs incubated with a fluorescent dye (FLIPR Membrane Potential Assay, Molecular Devices) that measures the change in membrane potential upon acetylcholine-induced nAChR activation. Protein shown (cyan-green) is α-elapitoxin-Dpp2d from D. polylepis (PDB ID: 4LFT). (f) The neurotoxic activity of Kunitz-type toxins on voltage-gated potassium channels will be measured in a cell-based assay using transfected HEK293 cells expressing human Kv1.1 or Kv1.2 channels. Cells are incubated with the fluorescent FLIPR Membrane Potential Assay dye (Molecular Devices) that measures the change in membrane potential upon application of extracellular potassium sulphate which causes cell membrane depolarisation and therefore activation of Kv channels. Protein shown (red-yellow) is dendrotoxin-I from D. polylepis (PDB ID: 1DEM).