ELISA and BLI analyses were performed as previously described (18 (link), 24 (link), 25 (link)). Briefly, ELISA plates coated with 2 µg/ml anti-His mAb were used to capture NFL trimers (2 µg/ml) followed by primary mAbs (five-fold serially diluted, starting from 10 µg/ml) and a peroxidase-conjugated goat anti-human secondary Ab (1:10,000). Plates were developed using 3,3′,5,5-tetramethylbenzidine chromagen solution. The data were plotted in GraphPad Prism version 7.
The BLI analyses were carried out on an Octet Red instrument (ForteBio) with IgGs immobilized on anti-human IgG Fc capture sensors (ForteBio). The NFL trimers were assessed as free analytes in solution (PBS pH 7.4). The analytes started from 200 nM and then twofold serially diluted to a final concentration of 12.5 nM. Association and dissociation times were 2 and 2 min or 3 and 3 min, respectively. Data were analyzed using the ForteBio analysis software version 7.1 (ForteBio) and the kinetic parameters were calculated using a global fit 1:1 model.
The BLI analyses were carried out on an Octet Red instrument (ForteBio) with IgGs immobilized on anti-human IgG Fc capture sensors (ForteBio). The NFL trimers were assessed as free analytes in solution (PBS pH 7.4). The analytes started from 200 nM and then twofold serially diluted to a final concentration of 12.5 nM. Association and dissociation times were 2 and 2 min or 3 and 3 min, respectively. Data were analyzed using the ForteBio analysis software version 7.1 (ForteBio) and the kinetic parameters were calculated using a global fit 1:1 model.
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