A Serological Snapshot of COVID-19 Initial Stages in Israel by a 6-Plex Antigen Array.

The first case of SARS-CoV-2 was discovered in Israel in late February 2020. Three major outbreaks followed, resulting in over 800,000 cases and over 6,000 deaths by April 2021. Our aim was characterization of a serological snapshot of Israeli patients and healthy adults in the early months of the COVID-19 pandemic. Sera from 55 symptomatic COVID-19 patients and 146 healthy subjects (early-pandemic, reverse transcription-quantitative PCR [qRT-PCR]-negative), collected in Israel between March and April 2020, were screened for SARS-CoV-2-specific IgG, IgM, and IgA antibodies, using a 6-plex antigen microarray presenting the whole inactivated virus and five viral antigens: a stabilized version of the spike ectodomain (S2P), spike subunit 1 (S1), receptor-binding-domain (RBD), N-terminal-domain (NTD), and nucleocapsid (NC). COVID-19 patients, 4 to 40 days post symptom onset, presented specific IgG to all of the viral antigens (6/6) in 54 of the 55 samples (98% sensitivity). Specific IgM and IgA antibodies for all six antigens were detected in only 10% (5/55) and 4% (2/55) of the patients, respectively, suggesting that specific IgG is a superior serological marker for COVID-19. None of the qRT-PCR-negative sera reacted with all six viral antigens (100% specificity), and 48% (70/146) were negative throughout the panel. Our findings confirm a low seroprevalence of anti-SARS-CoV-2 antibodies in the Israeli adult population prior to the COVID-19 outbreak. We further suggest that the presence of low-level cross-reacting antibodies in naive individuals calls for a combined, multiantigen analysis for accurate discrimination between naive and exposed individuals. IMPORTANCE A 6-plex protein array presenting the whole inactivated virus and five nucleocapsid and spike-derived SARS-CoV-2 antigens was used to generate a serological snapshot of SARS-CoV-2 seroprevalence and seroconversion in Israel in the early months of the pandemic. Our findings confirm a very low seroprevalence of anti-SARS-CoV-2 antibodies in the Israeli adult population. We further propose that the presence of low-level nonspecific antibodies in naive individuals calls for a combined, multiantigen analysis for accurate discrimination between naive and exposed individuals enabling accurate determination of seroconversion. The developed assay is currently applied to evaluate immune responses to the Israeli vaccine during human phase I/II trials.

An antigen microarray protocol for COVID-19 serological analysis.

The emergence of the coronavirus disease 2019 pandemic increased the interest in analysis of immunoglobulin responses. ELISA and lateral flow assays are widely used but are restricted by a single response value to an antigen or antigen pool. Here, we describe antigen microarrays, an alternative allowing simultaneous assessment of multiple interactions between antigens and the immunoglobulin content of patient sera. The technique requires minimal reagents and sample input and can be adapted to a wide variety of potential antigenic targets of interest.

Epitope-specific antibody responses differentiate COVID-19 outcomes and variants of concern.

BACKGROUNDThe role of humoral immunity in COVID-19 is not fully understood, owing, in large part, to the complexity of antibodies produced in response to the SARS-CoV-2 infection. There is a pressing need for serology tests to assess patient-specific antibody response and predict clinical outcome.METHODSUsing SARS-CoV-2 proteome and peptide microarrays, we screened 146 COVID-19 patients' plasma samples to identify antigens and epitopes. This enabled us to develop a master epitope array and an epitope-specific agglutination assay to gauge antibody responses systematically and with high resolution.RESULTSWe identified linear epitopes from the spike (S) and nucleocapsid (N) proteins and showed that the epitopes enabled higher resolution antibody profiling than the S or N protein antigen. Specifically, we found that antibody responses to the S-811-825, S-881-895, and N-156-170 epitopes negatively or positively correlated with clinical severity or patient survival. Moreover, we found that the P681H and S235F mutations associated with the coronavirus variant of concern B.1.1.7 altered the specificity of the corresponding epitopes.CONCLUSIONEpitope-resolved antibody testing not only affords a high-resolution alternative to conventional immunoassays to delineate the complex humoral immunity to SARS-CoV-2 and differentiate between neutralizing and non-neutralizing antibodies, but it also may potentially be used to predict clinical outcome. The epitope peptides can be readily modified to detect antibodies against variants of concern in both the peptide array and latex agglutination formats.FUNDINGOntario Research Fund (ORF) COVID-19 Rapid Research Fund, Toronto COVID-19 Action Fund, Western University, Lawson Health Research Institute, London Health Sciences Foundation, and Academic Medical Organization of Southwestern Ontario (AMOSO) Innovation Fund.

Multiplexed, microscale, microarray-based serological assay for antibodies against all human-relevant coronaviruses.

Rapid, sensitive, and precise multiplexed assays for serological analysis during candidate COVID-19 vaccine development would streamline clinical trials. The VaxArray Coronavirus (CoV) SeroAssay quantifies IgG antibody binding to 9 pandemic, potentially pandemic, and endemic human CoV spike antigens in 2 h with automated results analysis. IgG antibodies in serum bind to the CoV spike protein capture antigens printed in a microarray format and are labeled with a fluorescent anti-species IgG secondary label. The assay demonstrated excellent lower limits of quantification ranging from 0.3 to 2.0 ng/mL and linear dynamic ranges of 76 to 911-fold. Average precision of 11 % CV and accuracy (% recovery) of 92.5 % over all capture antigens were achieved over 216 replicates representing 3 days and 3 microarray lots. Clinical performance on 263 human serum samples (132 SARS-CoV-2 negatives and 131 positives based on donor-matched RT-PCR and/or date of collection) produced 98.5 % PPA and 100 % NPA.

Machine learning-based cytokine microarray digital immunoassay analysis.

Serial measurement of a large panel of protein biomarkers near the bedside could provide a promising pathway to transform the critical care of acutely ill patients. However, attaining the combination of high sensitivity and multiplexity with a short assay turnaround poses a formidable technological challenge. Here, the authors develop a rapid, accurate, and highly multiplexed microfluidic digital immunoassay by incorporating machine learning-based autonomous image analysis. The assay has achieved 12-plexed biomarker detection in sample volume <15 µL at concentrations < 5 pg/mL while only requiring a 5-min assay incubation, allowing for all processes from sampling to result to be completed within 40 min. The assay procedure applies both a spatial-spectral microfluidic encoding scheme and an image data analysis algorithm based on machine learning with a convolutional neural network (CNN) for pre-equilibrated single-molecule protein digital counting. This unique approach remarkably reduces errors facing the high-capacity multiplexing of digital immunoassay at low protein concentrations. Longitudinal data obtained for a panel of 12 serum cytokines in human patients receiving chimeric antigen receptor-T (CAR-T) cell therapy reveals the powerful biomarker profiling capability. The assay could also be deployed for near-real-time immune status monitoring of critically ill COVID-19 patients developing cytokine storm syndrome.

Analysis of SARS-CoV-2 antibodies in COVID-19 convalescent blood using a coronavirus antigen microarray.

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.

Evaluation of the Quotient® MosaiQ™ COVID-19 antibody microarray for the detection of IgG and IgM antibodies to SARS-CoV-2 virus in humans.

The MosaiQ® COVID-19 Antibody test fulfills the minimal requirements for serological testing according to the French regulation.

A modular microarray imaging system for highly specific COVID-19 antibody testing.

To detect the presence of antibodies in blood against SARS-CoV-2 in a highly sensitive and specific manner, here we describe a robust, inexpensive ($200), 3D-printable portable imaging platform (TinyArray imager) that can be deployed immediately in areas with minimal infrastructure to read coronavirus antigen microarrays (CoVAMs) that contain a panel of antigens from SARS-CoV-2, SARS-1, MERS, and other respiratory viruses. Application includes basic laboratories and makeshift field clinics where a few drops of blood from a finger prick could be rapidly tested in parallel for the presence of antibodies to SARS-CoV-2 with a test turnaround time of only 2-4 h. To evaluate our imaging device, we probed and imaged coronavirus microarrays with COVID-19-positive and negative sera and achieved a performance on par with a commercial microarray reader 100× more expensive than our imaging device. This work will enable large scale serosurveillance, which can play an important role in the months and years to come to implement efficient containment and mitigation measures, as well as help develop therapeutics and vaccines to treat and prevent the spread of COVID-19.