Development of a quantitative COVID-19 multiplex assay and its use for serological surveillance in a low SARS-CoV-2 incidence community.

Cassandra Guarino,Susanna Babasyan,Diego G Diel,Bettina Wagner,Elizabeth Plocharczyk,Melissa Laverack,Lara Parrilla,Alicia Rollins,Lok R Joshi,Elisabeth Larson,Gheyath K Nasrallah

doi:10.1371/journal.pone.0262868

Abstract

A serological COVID-19 Multiplex Assay was developed and validated using serum samples from convalescent patients and those collected prior to the 2020 pandemic. After initial testing of multiple potential antigens, the SARS-CoV-2 nucleocapsid protein (NP) and receptor-binding domain (RBD) of the spike protein were selected for the human COVID-19 Multiplex Assay. A comparison of synthesized and mammalian expressed RBD proteins revealed clear advantages of mammalian expression. Antibodies directed against NP strongly correlated with SARS-CoV-2 virus neutralization assay titers (rsp = 0.726), while anti-RBD correlation was moderate (rsp = 0.436). Pan-Ig, IgG, IgA, and IgM against NP and RBD antigens were evaluated on the validation sample sets. Detection of NP and RBD specific IgG and IgA had outstanding performance (AUC > 0.90) for distinguishing patients from controls, but the dynamic range of the IgG assay was substantially greater. The COVID-19 Multiplex Assay was utilized to identify seroprevalence to SARS-CoV-2 in people living in a low-incidence community in Ithaca, NY. Samples were taken from a cohort of healthy volunteers (n = 332) in early June 2020. Only two volunteers had a positive result on a COVID-19 PCR test performed prior to serum sampling. Serological testing revealed an exposure rate of at least 1.2% (NP) or as high as 5.7% (RBD), higher than the measured incidence rate of 0.16% in the county at that time. This highly sensitive and quantitative assay can be used for monitoring community exposure rates and duration of immune response following both infection and vaccination.

Highlights

With the goal to identify the best antigens for the COVID-19 Multiplex assay, different proteins of SARS-CoV-2 were evaluated and compared as antigen targets for a serological multiplex assay using a pan-Ig detection antibody
The assay based on synthetic receptorbinding domain (RBD) resulted in an area under the ROC curve (AUC) of 0.6496, indicating poor discrimination between patients and controls
The AUC for IL-4/RBD evaluated on the same sample set was 0.9945, indicating excellent discrimination between patients and controls

Summary

Introduction

The community of Ithaca, NY (in Tompkins county) responded swiftly and decisively to the impending threat of SARS-CoV-2, closing public and private schools, most child daycare. A comparison of three different assays on serum samples from a region of Japan with no identified SARS-CoV-2 outbreaks at the time testing was performed resulted in inconsistent seroprevalence values ranging from 0–3.3%, depending on which test was used [54] This highlights the importance for high sensitivity and specificity in order to accurately detect rare seropositive cases amongst the broad population. We demonstrate the importance of the antigen expression methodology for assay performance, compare assay results to virus neutralization, and evaluate the detection of different antibody isotypes We utilized this assay to Development of a COVID-19 Multiplex Assay and its use for surveillance in a low SARS-CoV-2 incidence community perform surveillance of the non-diagnosed exposure rate in the Cornell University community residing in Ithaca, NY in early June 2020

Materials and methods

Results

Discussion