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ELISA titration of mAb binding and fitting of EC50

ZK Zhiqiang Ku
XX Xuping Xie
ED Edgar Davidson
XY Xiaohua Ye
HS Hang Su
VM Vineet D. Menachery
YL Yize Li
ZY Zihao Yuan
XZ Xianwen Zhang
AM Antonio E. Muruato
AE Ariadna Grinyo i Escuer
BT Breanna Tyrell
KD Kyle Doolan
BD Benjamin J. Doranz
DW Daniel Wrapp
PB Paul F. Bates
JM Jason S. McLellan
SW Susan R. Weiss
NZ Ningyan Zhang
PS Pei-Yong Shi
ZA Zhiqiang An
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Corning high-binding assay plates were coated with recombinant sCoV2-RBD or sCoV-RBD protein (1 μg/ml) at 4 °C overnight and blocked with 5% skim milk at 37 °C for 2 h. Serially diluted antibodies were added at a volume of 100 μl per well for incubation at 37 °C for 2 h. The anti-human IgG Fab2 HRP-conjugated antibody was diluted 1:5000 and added at a volume of 100 μl per well for incubation at 37 °C for 1 h. The plates were washed 3–5 times with PBST (0.05% Tween-20) between incubation steps. TMB substrate was added 100 μl per well for color development for 3 min and 2 M H2SO4 was added 50 μl per well to stop the reaction. The OD 450 nm was read by a SpectraMax microplate reader. The data points were plotted by GraphPad Prism8, and the EC50 values were calculated using a three-parameter nonlinear model.

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