Single-domain antibodies against SARS-CoV-2 RBD from a two-stage phage screening of universal and focused synthetic libraries.

Publication date: Feb 13, 2024

Coronavirus disease 2019 (COVID-19) is an evolving global pandemic, and nanobodies, as well as other single-domain antibodies (sdAbs), have been recognized as a potential diagnostic and therapeutic tool for infectious diseases. High-throughput screening techniques such as phage display have been developed as an alternative to in vivo immunization for the discovery of antibody-like target-specific binders. We designed and constructed a highly diverse synthetic phage library sdAb-U (single-domain Antibody – Universal library ) based on a human framework. The SARS-CoV-2 receptor-binding domain (RBD) was expressed and purified. The universal library sdAb-U was panned against the RBD protein target for two rounds, followed by monoclonal phage ELISA (enzyme-linked immunosorbent assay) to identify RBD-specific binders (the first stage). High-affinity binders were sequenced and the obtained CDR1 and CDR2 sequences were combined with fully randomized CDR3 to construct a targeted (focused) phage library sdAb-RBD, for subsequent second-stage phage panning (also two rounds) and screening. Then, sequences with high single-to-background ratios in phage ELISA were selected for expression. The binding affinities of sdAbs to RBD were measured by an ELISA-based method. In addition, we conducted competition ELISA (using ACE2 ectodomain S19-D615) and SARS-CoV-2 pseudovirus neutralization assays for the high-affinity RBD-binding sdAb39. Significant enrichments were observed in both the first-stage (universal library) and the second-stage (focused library) phage panning. Five RBD-specific binders were identified in the first stage with high ELISA signal-to-background ratios. In the second stage, we observed a much higher possibility of finding RBD-specific clones in phage ELISA. Among 45 selected RBD-positive sequences, we found eight sdAbs can be well expressed, and five of them show high-affinity to RBD (EC 

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Concepts Keywords
Antibodies Phage display
Coronavirus RBD
Library SARS-CoV-2
Nanobodies Single-domain antibody
Stage Synthetic library


Type Source Name
disease MESH Coronavirus disease 2019
disease MESH infectious diseases
disease VO immunization
disease IDO assay
pathway REACTOME Reproduction
disease VO vaccine
drug DRUGBANK Spinosad
disease IDO production
disease VO storage
disease VO effective
disease MESH infection
disease MESH inflammation
disease MESH thrombocytopenic purpura
disease IDO host
disease VO Glycoprotein
drug DRUGBANK Angiotensin II
drug DRUGBANK Pirenzepine
disease VO efficient
disease IDO immune response
disease VO efficiency
drug DRUGBANK Amino acids
disease VO Bacteria
drug DRUGBANK Carbenicillin
drug DRUGBANK Dextrose unspecified form
drug DRUGBANK Polyethylene glycol
drug DRUGBANK Trestolone
drug DRUGBANK Trypsin
disease VO protocol
disease IDO colony
drug DRUGBANK Isopropyl beta-D-thiogalactopyranoside
drug DRUGBANK Esomeprazole
disease VO titer
drug DRUGBANK Flunarizine
drug DRUGBANK Sucrose
drug DRUGBANK Streptomycin
disease MESH dissociation
drug DRUGBANK Human Serum Albumin
disease VO Viruses
drug DRUGBANK Coenzyme M
drug DRUGBANK L-Arginine
drug DRUGBANK Methionine
drug DRUGBANK L-Cysteine
drug DRUGBANK L-Tryptophan
drug DRUGBANK L-Tyrosine
disease VO effectiveness
disease VO immunized
disease IDO process
disease VO organization
disease MESH Cancer
drug DRUGBANK (S)-Des-Me-Ampa
disease MESH Pneumonia
drug DRUGBANK Guanosine
drug DRUGBANK Indoleacetic acid
drug DRUGBANK Nonoxynol-9
disease MESH viral diseases
drug DRUGBANK Caplacizumab
disease IDO cell
disease VO inactivated vaccine
drug DRUGBANK Diethylstilbestrol

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