SARS-CoV-2 Serosurveys: How antigen, isotype and threshold choices affect the outcome (preprint)

Background: Evaluating the performance of SARS-CoV-2 serological assays and clearly articulating the utility of selected antigen, isotypes and thresholds is crucial to understanding the prevalence of infection within selected communities. Methods: This cross-sectional study, implemented in 2020, screened PCR-confirmed COVID-19 patients (n=86), banked pre-pandemic and negative donors (n=96), health care workers and family members (n=552), and university employees (n=327) for anti-SARS-CoV-2 receptor-binding domain (RBD), trimeric spike protein (S), and nucleocapsid protein (N) IgG and IgA antibodies with a laboratory developed Enzyme-Linked Immunosorbent Assay (ELISA) and tested how antigen, isotype and threshold choices affected the seroprevalence. The following threshold methods were evaluated: (i) mean + 3 standard deviations of the negative controls; (ii) 100% specificity for each antigen/isotype combination; and (iii) the maximal Youden index. Results: We found vastly different seroprevalence estimates depending on selected antigens, isotypes and the applied threshold method, ranging from 0.0% to 85.4%. Subsequently, we maximized specificity and reported a seroprevalence, based on more than one antigen, ranging from 9.3% to 25.9%. Conclusions: This study revealed the importance of evaluating serosurvey tools for antigen, isotype, and threshold-specific sensitivity and specificity, in order to interpret qualitative serosurvey outcomes reliably and consistently across studies.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization (preprint)

Background: In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and to develop, validate, improve, and implement serological testing and associated technologies. SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. Methods: To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. SARS-CoV-2 serology standard reference material and First WHO International Standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. Results: SeroNet institutions reported development of a total of 27 ELISA methods, 13 multiplex assays, 9 neutralization assays, and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. Conclusions: SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 virus and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons.