Antibody reactivity to HES and hydrogen fluoride (HF)-treated HES was performed as described previously (Hewitson et al., 2011 (link)). For competition ELISA, plate bound HES was first incubated for 30 min at 37 °C with 250 μg/ml of unlabelled mAb before addition of 10 μg/ml of biotin-labelled mAb for 2 h at 37 °C. Antibodies were biotinylated with a 20-fold molar excess of biotin using EZ-link Sulfo-NHS Biotinylation kit (Thermo Fisher Scientific, USA). Bound biotinylated antibody was detected with 1/1000 streptavidin-horseradish peroxidase (Sigma–Aldrich, USA) and developed as described by Hewitson et al. (2011) (link).
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