Binding kinetics were analyzed by BLI utilizing the Octet Red 96 system (ForteBio). All assays were conducted in standard Greiner black 96 microtiter well plates. Analyte (venom) samples were diluted 1:20 from the working stock to make a final experimental concentration of 50 µg/mL in the well (10 µg per well). Mimotope aliquots were diluted 1:50 to have a final concentration of 1 µg/mL in the well (0.2 µg per well). Assay running buffer was 1X DPBS with 0.1% BSA and 0.05% Tween-20. This buffer inhibits non-specific binding to the surface of the sensor and other proteins. Prior to experimentation, streptavidin sensors were hydrated in the running buffer for 30–60 min, whilst being agitated at 2.0 RPM on a shaker. To regenerate the sensor tips during experimentation, the dissociation of analytes occurs using a standard acidic solution (glycine buffer), made up of 10mM glycine (pH 1.5–1.7) in deionized water.
Octet RED 96 assay methodology in the ForteBio Data Acquisition 9.0 program was set as follows: 60 s baseline, 50 s loading, 120 s baseline, 120 s association, 120 s dissociation, and 80 s regeneration/neutralization step. The regeneration/neutralization step consists of four cycles, lasting 10 s each, alternating between dipping in glycine buffer (regeneration) and then in running buffer (neutralization) per cycle. For each baseline step throughout the experiment the same running buffer was used a maximum of three times per well. Experiments were run at 30 °C with the orbital agitation of the microplate set to 1000 rpm. Experiments were limited to less than 1 h to limit the change in analyte concentration due to evaporation on the warmed plate [32 (link)].
Analytes were set up in rows A-H, with triplicates set up in columns 1–3 and 4–6. To account for any potential evaporation effect in the wells during experimentation, the column running order was set to 1, 4, 2, 5, 3, 6 (rather than 1, 2, 3, 4, 5, 6). The mimotopes were set in column 7, running buffer was set in columns 8–10, and regeneration step (glycine buffer) and neutralization step (running buffer) were columns 11 and 12 respectively.
Negative controls consisted of deionized water:glycerol 1:1 mix in replacement of the sample in the wells. Bungarus multicinctus venom was used as a positive control, as it has been shown to bind nAChR mimotopes [14 (link),15 (link),16 (link)].
Octet RED 96 assay methodology in the ForteBio Data Acquisition 9.0 program was set as follows: 60 s baseline, 50 s loading, 120 s baseline, 120 s association, 120 s dissociation, and 80 s regeneration/neutralization step. The regeneration/neutralization step consists of four cycles, lasting 10 s each, alternating between dipping in glycine buffer (regeneration) and then in running buffer (neutralization) per cycle. For each baseline step throughout the experiment the same running buffer was used a maximum of three times per well. Experiments were run at 30 °C with the orbital agitation of the microplate set to 1000 rpm. Experiments were limited to less than 1 h to limit the change in analyte concentration due to evaporation on the warmed plate [32 (link)].
Analytes were set up in rows A-H, with triplicates set up in columns 1–3 and 4–6. To account for any potential evaporation effect in the wells during experimentation, the column running order was set to 1, 4, 2, 5, 3, 6 (rather than 1, 2, 3, 4, 5, 6). The mimotopes were set in column 7, running buffer was set in columns 8–10, and regeneration step (glycine buffer) and neutralization step (running buffer) were columns 11 and 12 respectively.
Negative controls consisted of deionized water:glycerol 1:1 mix in replacement of the sample in the wells. Bungarus multicinctus venom was used as a positive control, as it has been shown to bind nAChR mimotopes [14 (link),15 (link),16 (link)].
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