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

• Published in: BMC infectious diseases 2024-02, Vol.24 (1), p.199-199, Article 199
• Main Authors: Chen, Fangfang, Liu, Zhihong, Kang, Wei, Jiang, Fan, Yang, Xixiao, Yin, Feng, Zhou, Ziyuan, Li, Zigang
• Format: Article
• Language: English
• Subjects: ACE2; Affinity; Analysis; Angiotensin-Converting Enzyme 2; Antibodies; Antibodies, Neutralizing; Antibodies, Viral; Antigens; Bacteriophages; Binders; Binding; Camelids; Cloning; Complementarity-determining region 1; Complementarity-determining region 3; Coronaviruses; COVID-19; COVID-19 - diagnosis; COVID-19 vaccines; DNA polymerase; E coli; Enzyme-linked immunosorbent assay; Glycoproteins; High-throughput screening; Humans; Identification and classification; Immunization; Immunoglobulins; In vivo methods and tests; Infectious diseases; Libraries; Medical research; Nanobodies; Neutralization; Pandemics; Panning; Phage display; Phages; Production costs; RBD; SARS-CoV-2; SARS-CoV-2 - genetics; Severe acute respiratory syndrome coronavirus 2; Single-Domain Antibodies - chemistry; Single-Domain Antibodies - genetics; Single-domain antibody; Strategy; Synthetic library; Testing; Viral antibodies; Viral diseases
• ISSN: 1471-2334; 1471-2334
• DOI: 10.1186/s12879-024-09022-8

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