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AlphaScreen assay

YS Yukako Senga
HI Hiroshi Imamura
TM Takamitsu Miyafusa
HW Hideki Watanabe
SH Shinya Honda
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The assay was performed in a white 384-well OptiPlate (PerkinElmer, Waltham, MA, USA) containing 4 μM biotin-AF.2A1, 10 μg/ml acceptor beads conjugated to AF.2A1, and non-native IgG in AlphaScreen buffer (PBS, 1% Tween 20, and 1% bovine serum albumin). Reaction mixtures were incubated at 23 °C for 60 min. Streptavidin donor beads (20 μl at 100 μg/ml) were added (also in AlphaScreen buffer), and the plate was incubated at 23 °C for 30 min in the dark. Next, it was read on an EnSpire™ Alpha (PerkinElmer, Waltham, MA, USA) using the AlphaScreen protocol. LOD and LOQ were calculated from the calibration curve as 3.3 s/slope and 10 s/slope, respectively, where s is the standard deviation of the blank signal.

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