Validation and performance of a multiplex serology assay to quantify antibody responses following SARS-CoV-2 infection or vaccination

Abstract

Objectives: Robust, quantitative serology assays are required to accurately measure antibody levels following vaccination and natural infection. We present validation of a quantitative, multiplex, SARS-CoV-2, electrochemiluminescent (ECL) serology assay; show correlation with two established SARS-CoV-2 immunoassays; and present calibration results for two SARS-CoV-2 reference standards.

Methods: Precision, dilutional linearity, ruggedness, analytical sensitivity and specificity were evaluated. Clinical sensitivity and specificity were assessed using serum from prepandemic and SARS-CoV-2 polymerase chain reaction (PCR)-positive patient samples. Assay concordance to the established Roche Elecsys® Anti-SARS-CoV-2 immunoassay and a live-virus microneutralisation (MN) assay was evaluated.

Results: Standard curves demonstrated the assay can quantify SARS-CoV-2 antibody levels over a broad range. Assay precision (10.2-15.1% variability), dilutional linearity (≤ 1.16-fold bias per 10-fold increase in dilution), ruggedness (0.89-1.18 overall fold difference), relative accuracy (107-118%) and robust selectivity (102-104%) were demonstrated. Analytical sensitivity was 7, 13 and 7 arbitrary units mL^-1 for SARS-CoV-2 spike (S), receptor-binding domain (RBD) and nucleocapsid (N) antigens, respectively. For all antigens, analytical specificity was > 90% and clinical specificity was 99.0%. Clinical sensitivities for S, RBD and N antigens were 100%, 98.8% and 84.9%, respectively. Comparison with the Elecsys® immunoassay showed ≥ 87.7% agreement and linear correlation (Pearson r of 0.85, P < 0.0001) relative to the MN assay. Conversion factors for the WHO International Standard and Meso Scale Discovery® Reference Standard are presented.

Conclusions: The multiplex SARS-CoV-2 ECL serology assay is suitable for efficient, reproducible measurement of antibodies to SARS-CoV-2 antigens in human sera, supporting its use in clinical trials and sero-epidemiology studies.

Keywords: COVID‐19; SARS‐CoV‐2; antibodies; electrochemiluminescence; immunoassay; serology.

Figure 1

Standard curve precision profiles for SARS‐CoV‐2‐specific S, RBD and N antibodies 11‐point dilution series tested 45 times. AU, arbitrary units; LOQ, limit of quantitation; N, nucleocapsid protein; RBD, receptor‐binding domain; RLU, relative light unit; S, spike protein; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.

Figure 2

Analytical specificity (a) and sensitivity (b) of the multiplex SARS‐CoV‐2 ECL serology assay to measure antibodies to heterologous and homologous antigens. ECL, electrochemiluminescence; H3, H3 Hong Kong influenza hemagglutinin; N, nucleocapsid protein; OC43, seasonal coronavirus OC43 spike protein; RBD, receptor‐binding domain; S, spike protein; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.

Figure 3

SARS‐CoV‐2 S, RBD and N antibody distribution by serostatus cut points (dashed lines) in samples from donors according to known SARS‐CoV‐2 status (a) and number of PCR‐positive samples that were seronegative versus seropositive for both S and N (b) ^†One measurement failed run validity criteria. In a, GMT values for each group are shown as solid‐coloured lines. In b, RBD versus N distribution is the same as S versus N and is not shown. AU, arbitrary units; ECL, electrochemiluminescence; GMT, geometric mean titre; N, nucleocapsid protein; PCR, polymerase chain reaction; RBD, receptor‐binding domain; S, spike protein; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.

Figure 4

SARS‐CoV‐2 S, RBD and N antibody distribution by serostatus cut points (dashed lines) in samples from donors with known Roche Elecsys® Anti‐SARS‐CoV‐2 immunoassay results (a) and distribution of S versus N antibody concentrations as measured by the multiplex SARS‐CoV‐2 ECL serology assay (b). In a, GMT values for each group are shown as solid‐coloured lines. In b, RBD versus N distribution is the same as S versus N and is not shown. AU, arbitrary units; ECL, electrochemiluminescence; GMT, geometric mean titre; N, nucleocapsid protein; RBD, receptor‐binding domain; S, spike protein; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.

Figure 5

Correlation between the multiplex SARS‐CoV‐2 ECL serology assay (S assay) and the BBRC SARS‐CoV‐2 live‐virus MN assay. A total of 57 samples were tested in three assays. Horizontal dotted line indicates the multiplex SARS‐CoV‐2 ECL serology S assay cut point (675 AU mL⁻¹). Vertical dotted line indicates BBRC MN assay LLOQ (IC₅₀ = 20). Colour shows Roche Elecsys® Anti‐SARS‐CoV‐2 immunoassay results. AU, arbitrary units; BBRC, Battelle Biomedical Research Center; CI, confidence interval; ECL, electrochemiluminescence; IC₅₀, half maximal inhibitory concentration; LLOQ, lower limit of quantitation; MN, microneutralisation; S, spike protein; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.