Biopanning of Peptides Specific to the Conserved Epitope of the SARS-CoV-2 Receptor-Binding Domain and Nanozyme-Assisted Dual Peptide-Based Sandwich ELISA for Ultrasensitive Detection of the SARS-CoV-2 Antigen.
TL;DR
This study proposed phage-displayed dual peptides to construct a reliable and inexpensive ELISA for screening SARS-CoV-2 infection and found the results are well consistent with those of the gold-standard RT-PCR method.
Abstract
As the subunit of the SARS-CoV-2 spike protein (SARS-CoV-2 SP), the receptor-binding domain (RBD) can specifically bind to human angiotensin-converting enzyme 2 (ACE2), enabling viral infection of host cells. Therefore, it is of utmost significance to explore probes that can bind to the conserved epitope of the SARS-CoV-2 RBD with good affinity, so as to recognize various SARS-CoV-2 variants for diagnosis, vaccination, and the development of new detection methods. Herein, we biopanned peptide RN3 (sequence: YSIDWVFHHPML) by phage display, which can bind to the SARS-CoV-2 RBD with excellent affinity and selectivity. Molecular dynamics simulation and molecular docking verified that the His8 and Met11 residues in peptide RN3 are bound to the Asn487 and Tyr489 residues of the conserved epitope of the SARS-CoV-2 RBD subunit on the SARS-CoV-2 SP (His8-Asn487 and Met11-Tyr489, respectively) by hydrogen bonding. Alanine scanning confirmed that Met11 of peptide RN3 is the key amino acid for binding to the SARS-CoV-2 SP. The median inhibition concentration for peptide RN3 inhibiting the SARS-CoV-2 SP binding to ACE2 was 37 nM. Subsequently, phage RN3 was used as a capture probe, the SARS-CoV-2 SP-specific binding peptide Pn (sequence: WNLDLSQWLPPMGGGSKKKC) as a detection probe, and Au@Pd NP-based peroxidase-mimicking nanozyme for signal amplification. A phage RN3/antigen/peptide Pn sandwich ELISA was established for the selective detection of SARS-CoV-2 SP with a linear range of 5-1000 pg/mL and a limit of detection of 2.93 pg/mL. This method was applied to double-blind testing of 13 randomized clinical samples, which could distinguish between positive and negative samples. The detected results are well consistent with those of the gold-standard RT-PCR method. Thus, this study proposed phage-displayed dual peptides to construct a reliable and inexpensive ELISA for screening SARS-CoV-2 infection. A similar strategy can be extended to study other pathogens.