Human monoclonal antibodies block the binding of SARS-CoV-2 spike protein to angiotensin converting enzyme 2 receptor

Lilin Ye; Xiangyu Chen; Ren Li; Zhiwei Pan; Chunfeng Qian; Yang Yang; Renrong You; Jing Zhao; Leiqong Gao; Zhirong Li; Qizhao Huang; Lifan Xu; Jianfang Tang; Qin Tian; Wei Yao; Li Hu; Xiaofeng Yan; Xinyuan Zhou; Pinghuang Liu; Yuzhang Wu; Kai Deng; Zheng Zhang; Yaokai Chen; Zhaohui Qian

The copyright holder for this preprint (which was not peer-reviewed) is Biological,!10108^H08H)!in!100!μl!PBS!per!well!was!coated!on!ELISA!plates!overnight! at!4 o C.!Then,!the!ELISA!plates!were!blocked!for!1!hour!with!blocking!buffer!(5%!FBS! plus! 0.05%! Tween! 20)o! meanwhile,! three^fold! serial! dilutions! of! mAbs! or! ten^fold! diluted!patient!sera!were!incubated!with!optimal!dose!(based!on!EC 50 )!of!SARS^Cov2! RBD!protein!(Sino!Biological,!40592^V05H)!for!1!hour.!Then,!the!incubated!mixtures! were! added! to! ELISA! plates! and! allowed! to! develop! for! 30! min,! followed! by! PBST! washing!and!anti^mouse!Fc!HRP!antibody!(Thermo!Fisher!Scientific).!Next,!the!ELISA! plates! were! washed! with! PBST,! then! PBS! and! added! with! TMB! (Beyotime).! After! 5! min,! the! ELISA! plates! were! stopped! and! determined! at! 450! nm.! The! half! maximal! inhibitory! concentration! (IC 50 )! was! determined! by! using! 4^parameter! logistic! regression.& & Flow& cytometry7based& receptor7binding& inhibition& assay.& 311mab! mAbs! or! isotype! were! incubated! with! optimal! dose! (based! on! EC 50 )! of! SARS^Cov2! RBD! protein! (Sino! Biological,! 40592^V05H)! for! 1! hour! at! RT.! Then,! the! mixtures! were! incubated!with!10,000!hACE2^plasmid!transiently!transfected!293T!cells!for!40!min!on! ice,! followed! by! stained! with! Alexa! Fluor! 647^conjugated! goat! anti^mouse! IgG! (Biolegend)!and!APC^Cy7^conjugated!LIVE/DEAD!dye!(Life!Technologies).& & Pseudovirus&neutralization&assay.!Spike!protein!of!SARS^Cov^2!typed!pseudovirus! was!produced!as!previously!described 22 .!Concisely,!HEK^293T!cells!were!transfected! with! psPAX2,! pLenti^GFP! and! 2019^nCov! S! plasmids! by! using! TransIT^293! All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.