BIOENGINEERED | VOL 13 | NO 5 | 21 MAY 2022

Nileena Velappan, Hau B. Nguyen, Sofiya Micheva-Viteva, Daniel Bedinger, Chunyan Ye, Betty Mangadu, Austin J. Watts, Robert Meagher, Steven Bradfute, Bin Hu, Geoffrey S. Waldo, and Antonietta M. Lillo


Here, we describe the isolation of 18 unique anti SARS-CoV-2 human single-chain antibodies from an antibody library derived from healthy donors. The selection used a combination of phage and yeast display technologies and included counter-selection strategies meant to direct the selection of the receptor-binding motif (RBM) of SARS-CoV-2 spike protein’s receptor binding domain (RBD2). Selected antibodies were characterized in various formats including IgG, using flow cytometry, ELISA, high throughput SPR, and fluorescence microscopy. We report antibodies’ RBD2 recognition specificity, binding affinity, and epitope diversity, as well as ability to block RBD2 binding to the human receptor angiotensin-converting enzyme 2 (ACE2) and to neutralize authentic SARS-CoV-2 virus infection in vitro. We present evidence supporting that: 1) most of our antibodies (16 out of 18) selectively recognize RBD2; 2) the best performing 8 antibodies target eight different epitopes of RBD2; 3) one of the pairs tested in sandwich assays detects RBD2 with sub-picomolar sensitivity; and 4) two antibody pairs inhibit SARS-CoV-2 infection at low nanomolar half neutralization titers. Based on these results, we conclude that our antibodies have high potential for therapeutic and diagnostic applications. Importantly, our results indicate that readily available non immune (naïve) antibody libraries obtained from healthy donors can be used to select high-quality monoclonal antibodies, bypassing the need for blood of infected patients, and offering a widely accessible and low-cost alternative to more sophisticated and expensive antibody selection approaches (e.g. single B cell analysis and natural evolution in humanized mice).


  • Selected 16 antibodies specific for RBD2 and 2 cross reacting with RBD1
  • Affinities of 8 best-performing antibodies for RBD2 range from 27 to 800 nM
  • All best performers appear to target a different epitope of RBD and form 18 pairs
  • E01, F07, and S01 neutralize viral infection at low nanomolar NT50 when paired up
  • Pair F07+S01 can detect spike protein at LoD 160 fM


The world is currently experiencing a pandemic of coronavirus disease 2019 (COVID-19), caused by a novel severe acute respiratory syndrome (SARS)- like coronavirus (SARS-CoV-2). This is the seventh known coronavirus to infect humans, and the third causing widespread diseases, following SARS-CoV -1 12599-1 and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) [1–4]. SARS-CoV-2 is 79% identical and 86.1% homologous to SARS- CoV-1 and is classified into the genus betacoronavirus in the family Coronaviridae [2].

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