Evaluation of spike protein antigens for SARS-CoV-2 serology

Abstract

Background: Spike protein domains are being used in various serology-based assays to detect prior exposure to SARS-CoV-2 virus. However, there has been limited comparison of antibody titers against various spike protein antigens among COVID-19 infected patients.

Methods: We compared four spike proteins (RBD, S1, S2 and a stabilized spike trimer (ST)) representing commonly used antigens for their reactivity to human IgG antibodies using indirect ELISA in serum from COVID-19 patients and pre-2020 samples. ST ELISA was also compared against the EUROIMMUN IgG ELISA test. Further, we estimated time appropriate IgG and IgA seropositivity rates in COVID-19 patients using a panel of sera samples collected longitudinally from the day of onset of symptoms (DOS).

Results: Among the four spike antigens tested, the ST demonstrated the highest sensitivity (86.2 %; 95 % CI: 77.8-91.7 %), while all four antigens showed high specificity to COVID-19 sera (94.7-96.8 %). 13.8 % (13/94) of the samples did not show seroconversion in any of the four antigen-based assays. In a double-blinded head-to-head comparison, ST based IgG ELISA displayed a better sensitivity (87.5 %, 95 % CI: 76.4-93.8 %) than the EUROIMMUN IgG ELISA (67.9 %, 95 % CI: 54.8-78.6 %). Further, in ST-based assays, we found 48 % and 50 % seroconversion in the first six days (from DOS) for IgG and IgA antibodies, respectively, which increased to 84 % (IgG) and 85 % (IgA) for samples collected ≥22 days from DOS.

Conclusions: Comparison of spike antigens demonstrates that spike trimer protein is a superior option as an ELISA antigen for COVID-19 serology.

Keywords: SARS-CoV-2; Serology; Spike trimer ELISA.

Fig. 1

Reactivity of COVID-19 positive (n = 94 samples collected ≥15 days from day of -onset of symptoms or RT-PCR positivity) and control sera (n = 94) to different antigens (a) Trimeric prefusion spike protein structure (PDB: 5XLR (Gui et al., 2017)) shows the antigens used in the ELISA (red: RBD, yellow and red: S1 domain, blue: S2 domain, two monomers are represented in white) (b) Corrected OD (450 nm) value for S1, S2, RBD and ST protein for each sample is represented by a point on the scatter plot. Median, 5th percentile and 95th percentile values are shown by horizontal grey lines. Dotted lines indicate the cut-off values. (mean + 3 x standard deviation of corrected OD values of control samples). (c) Correlation between the four antigens. Corrected OD values for each antigen are plotted against the values for the other antigens. Pearson’s correlation coefficient for each pair of antigens is shown on the scatter plots. (d) Receiver operating characteristic (ROC) curve for each antigen.

Fig. 2

Head-to-head double-blinded comparison of EUROIMMUN Anti-SARS-CoV-2 ELISA (IgG) kit and ST ELISA. COVID-19 positive samples (filled circles) and control samples (empty circles) are plotted and Pearson’s correlation coefficient (PCC) between the two test values is indicated for the COVID-19 positive samples.

Fig. 3

Dynamics of ST protein-specific IgG and IgA response. (a-b) Corrected OD values of COVID-19 positive samples collected at different time points post-onset of symptoms (n = 25 for each time interval). Value for each sample is represented by a point on the scatter plot. Median, 5th percentile and 95th percentile values are shown by horizontal grey lines. (c) Percentage seroconversion of patients based on IgG and IgA response to ST protein ELISA as a function of time. Vertical lines show 95 % confidence intervals (d) Comparison between ranks of the IgG and IgA corrected OD values.