SARS-CoV-2 Serosurveys: How Antigen, Isotype and Threshold Choices Affect the Outcome

Abstract

Background: Evaluating the performance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays and clearly articulating the utility of selected antigens, isotypes, and thresholds is crucial to understanding the prevalence of infection within selected communities.

Methods: This cross-sectional study, implemented in 2020, screened PCRconfirmed coronavirus disease 2019 patients (n 86), banked prepandemic and negative samples (n 96), healthcare workers and family members (n 552), and university employees (n 327) for antiSARS-CoV-2 receptor-binding domain, trimeric spike protein, and nucleocapsid protein immunoglobulin (Ig)G and IgA antibodies with a laboratory-developed enzyme-linked immunosorbent assay and tested how antigen, isotype and threshold choices affected the seroprevalence outcomes. 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 and 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, to interpret qualitative serosurvey outcomes reliably and consistently across studies.

Keywords: ELISA; Massachusetts; SARS-CoV-2; Youden index; antigen; isotype; serosurvey; threshold.

Figure 1.

Optical density (OD) value distribution among each measured subgroup. OD values (450-570nm) were plotted according to each antigen-isotype combination and subgroup (y-axis in log10 scale). The top horizontal line in the graph indicates the cut off for the 3 standard deviation above the mean threshold method (3 SD). The second line indicates the cut off when the threshold was chosen at the highest value of the negative controls (Max Spec). The third line indicates the cut off when the threshold was chosen based on the Youden threshold (Youden). Note, the last two lines converge for S IgA and S IgG. Abbreviations: CHC Neg, PCR-negative CHCs (COVID-19 Hospitalized Case); CHC Pos, PCR confirmed hospitalized COVID-19 patients; HCW, health care workers; HCW Family, family members of health care worker listed under HCW; N, SARS-CoV-2 nucleocapsid protein; Neg, negative controls; RBD, SARS-CoV-2 receptor-binding domain; RTW, return to work employees who had been working remotely from March to August of 2020, during the first COVID-19 wave in Worcester, MA; S Trimer; SARS-CoV-2 spike trimer.

Figure 2.

Heat map of optical density (OD) values. A heat map was built based on unbiased clustering of the OD values of the experimental subgroups with at least one positive antigen-isotype combination. The OD values are represented in the color scale, ranging from blue to yellow. The results clustered by isotype, rather than antigen (see top branching into immunoglobulin [Ig]G and IgA from left to the right). Abbreviations: CHC, COVID-19 hospitalized case; HCW, healthcare workers and their family members; N, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein; RBD, SARS-CoV-2 receptor-binding domain; RTW, return-to-work employees who had been working remotely from March to August 2020, during the first COVID-19 wave in Worcester, Massachusetts; S trimer, SARS-CoV-2 spike trimer.

Figure 3.

Seropositivity of each subgroup, according to the antigen-isotype combination. The red bars represent the seroprevalence based on the threshold defined as the mean + 3 SDs among negative controls (3 SD). The blue bars represent the seroprevalence based on the maximum specificity threshold (Max Spec). The black bars represent the seroprevalence based on the Youden threshold (Youden). Abbreviations: HCWs, healthcare workers; HCW Family , family members of participating HCWs; Ig, immunoglobulin; N, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein; RBD, SARS-CoV-2 receptor-binding domain; RTW, return-to-work employees who had been working remotely from March to August of 2020, during the first coronavirus disease 2019 wave in Worcester, Massachusetts; S trimer, SARS-CoV-2 spike trimer; Top Three, combination of the three antigen/isotype combinations (RBD IgG, N IgG, and RBD IgA) with the largest area under the ROC curve (i.e., with maximum sensitivity and specificity).