Simultaneous measurement of the antibody responses against SARS-CoV-2 and its multiple variants by a phage display mediated immuno-multiplex quantitative PCR-based assay

Abstract

To combat the continued pandemic of COVID-19, multiplex serological assays have been developed to comprehensively monitor the humoral immune response and help to design new vaccination protocols to different SARS-CoV-2 variants. However, multiplex beads and stably transfected cell lines require stringent production and storage conditions, and assays based on flow cytometry is time-consuming and its application is therefore restricted. Here, we describe a phage display system to distinguish the differences of immune response to antigenic domains of multiple SARS-CoV-2 variants simultaneously. Compared with linear peptides, the recombinant antigens displayed on the phage surface have shown some function that requires the correct folding to form a stable structure, and the binding efficiency between the recombinant phage and existing antibodies is reduced by mutations on antigens known to be important for antigen–antibody interaction. By using Phage display mediated immuno-multiplex quantitative PCR (Pi-mqPCR), the binding efficiency between the antibody and antigens of different SARS-CoV-2 variants can be measured in one amplification reaction. Overall, these data show that this assay is a valuable tool to evaluate the humoral response to the same antigen of different SARS-CoV-2 variants or antigens of different pathogens. Combined with high-throughput DNA sequencing technology, this phage display system can be further applied in monitoring humoral immune response in a large population before and after vaccination.

Studying SARS-CoV-2 interactions using phage-displayed receptor binding domain as a model protein

“…Cross-inhibition shown with the phage-based ELISA, as well as the moderate loss of inhibitory power against mutated RBD variants (particularly Beta RBD) were subsequently confirmed by classical neutralization assays 47 . Phage display was subsequently used by other authors to study humoral responses to mutated RBD variants, but their approach differed from the current one in display multivalency (RBD proteins were fused to all copies of PIII due to gene insertion into the phage genome) and in the assay format (phage display mediated immuno-multiplex quantitative PCR) 48 .…”

A Competitive Panning Method Reveals an Anti-SARS-CoV-2 Nanobody Specific for an RBD-ACE2 Binding Site

“…Using the antibody detection method based on phage display technology that was successfully constructed in our laboratory previously [ 25 ], the binding activity of the screened VHH 5-05 to the recombinant phage displaying the SARS-CoV-2 mutant RBD was preliminarily verified. The recombinant M13KO7 phage containing the RBD fragments of each mutant strain of SARS-CoV-2 was prepared, and purified VHH 5-05 was coated on a high-adsorption microplate overnight at 4 °C.…”

Massively-multiplexed epitope mapping techniques for viral antigen discovery

“…Phage display libraries have been made up to 10 11 pfu/ml in size and can contain thousands of different peptides/proteins ( 64 ). Indeed, bacteriophages have been used to display functional SARS-Cov-2 Spike RBDs on the surface, coupled to functional screening, indicating a role for structural conformation of whole protein domains present on the phage surface ( 65 ). Additionally, phage-based protein scaffold libraries have already been developed for surface presentation of small proteins ( 66 ).…”