ReScan, a Multiplex Diagnostic Pipeline, Pans Human Sera for SARS-CoV-2 Antigens

Abstract

Word Count: 98 Abstract 55 Serologic assays are needed to determine SARS-CoV-2 seroprevalence, but poor specificity can overestimate exposures. 56Here, we built a pan-human coronavirus proteome-wide programmable phage display assay (VirScan) to profile 57 coronavirus antigens specifically enriched by 20 COVID-19 patient serum IgG. With ReScan, a new diagnostic 58 development workflow which combines the isolation of phage expressing the most immunogenic peptides with paper-59 based microarrays manufactured via acoustic liquid handling, we identified 9 candidate antigens from a library of 534 60 SARS-CoV-2 peptides. These arrays could form the basis of a multiplexed COVID-19 serologic assay with enhanced 61 specificity. ReScan has broad applicability for serologic assay development. 62 63 Article (2,000-4,000 words excluding abstract, online methods, refs, figure legends) 64 65

Systematic profiling of SARS-CoV-2 specific IgG epitopes at single amino acid resolution

“…These results are highly consistent to that of the S protein peptide microarray that we constructed (15). According to a SARS-CoV-2 specific peptide library based study (19), the highly immunogenic regions of Spike protein were also enriched in CTD, region around S2' and the fusion peptide, and HR2, which are highly consistent to our study. While aa362-399 of N protein which identified as high immunogenic region was also determined in our study, but other highly immunogenic regions were not.…”

“…There is great interest to profile pathogen specific IgG responses by using serum or plasma because of the convenience of sample collection. Protein microarray could be applied to reveal the pathogen specific IgG responses at protein level (16,33), while peptide microarray (15) or pathogen specific peptide library (19,22) were able to provide information at peptide/epitope level. However, these platforms could not provide epitope information at amino acid level, and pathogen specific protein or peptide library need to be constructed individually, which is costly and time-consuming.…”

“…By analyzing sera from 1,051 patients on a S protein peptide microarray of full S protein coverage, several areas and a set of peptides of high IgG reactivity were identified (14). Similar studies were also performed by using another peptide microarray (17), and a coronavirus specific phage displayed peptide library (19). The IgG responses on protein and peptide levels, but not single amino acid level, were at least partially revealed by these studies.…”

Engineering luminescent biosensors for point-of-care SARS-CoV-2 antibody detection

“…To construct the N sensors, we used the N-terminal sequence because aa 44–257 are found to be more immunogenic than the C-terminal dimerization domain (aa 258–419) ( Figure S5A ) ( Zamecnik et al, 2020 ). In addition, dimerization promoted by the C-terminal domain may lead to high basal NanoLuc reconstitution levels.…”

From multiplex serology to serolomics – a novel approach to the antibody response against the SARS-CoV-2 proteome

“…Thus far, only a few studies have employed either microarray or fluorescent-bead based technologies to develop multiplex SARS-CoV-2 serological assays (Becker et al, 2020;Dobaño et al, 2020;Jiang et al, 2020;Roxhed et al, 2020;Zamecnik et al, 2020;Zhang et al, 2020), all providing high specificity and sensitivity in detecting SARS-CoV-2 antibodies by varying combinations of proteins N and S, as well as subdomains or peptides thereof. Microarraybased studies utilized peptides or proteins of the SARS-CoV-2 proteome (Jiang et al, 2020;Zamecnik et al, 2020;Zhang et al, 2020) allowing for assessment of the immunogenicity of proteins other than N and S. In contrast to fluorescent-bead based technologies, microarraybased assays are, however, not suited for high-throughput analyses of large sample sets.…”