Labelled proteins were diluted to a final concentration of 5 μg/ml in PBS and coated onto the wells of a 96-well polystyrene plate (Pierce, ThermoFisher Scientific, Waltham, MA) by incubating overnight at 4°C. The plate was washed three times with PBST (PBS with 0.1% Tween-20) followed by blocking with 5% skim milk in PBST for 3h at room temperature. The blocking solution was removed, and the plate was again washed twice with PBST. Proteins were probed with KZ52 antibody [26 (link),35 (link)] at a dilution of 1:1000 overnight at 4°C. The plate was then washed and incubated with horseradish peroxidase-conjugated anti-human IgG (Invitrogen, ThermoFisher Scientific, Waltham, MA) at a dilution of 1:2000 for 2h at room temperature. After washing the plate four times with PBST, TMB solution (3,3’,5,5’-tetramethylbenzidine; ThermoFisher Scientific, Waltham, MA) was added to each well, incubated for 15 min, followed by addition of an equal volume of 2M sulphuric acid. The optical density was immediately read at 450 nm in a Synergy H1 microplate reader (BioTek, Winooski, VT).
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