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

Abstract

Abstract A serological COVID-19 Multiplex Assay was developed and validated using serum sample sets from convalescent patients and those collected prior to the onset of the 2020 pandemic. After initial testing of multiple potential antigens expressed in a mammalian system, the SARS-CoV-2 nucleoprotein (NP) and receptor-binding domain (RBD) of the spike protein were selected for the final human COVID-19 Multiplex Assay. A comparison of synthesized and mammalian expressed RBD proteins revealed clear advantages of the mammalian protein. Antibodies directed against NP strongly correlated with SARS-CoV-2 virus neutralization assay titers (rsp = 0.726), while the anti-RBD correlation was moderate (rsp = 0.436). Pan-Ig, IgG, IgA, and IgM against the 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 than for IgA. The COVID-19 Multiplex Assay was then 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 5.1% (RBD), which was higher than the measured incidence rate of 0.16% in the county at that time. This highly sensitive and quantitative assay will be useful for continuing to monitor community exposure rates, duration of immune response following infection, and monitoring vaccination responses as vaccines become widely available.