Two-tiered SARS-CoV-2 seroconversion screening in the Netherlands and stability of nucleocapsid, spike protein domain 1 and neutralizing antibodies

Abstract

Background: Serological testing in the COVID-19 pandemic is mainly implemented to gain sero-epidemiological data, but can also retrospectively inform about suspected SARS-CoV-2 infection.

Method: We verified and applied a two-tiered testing strategy combining a SARS-CoV-2 receptor-binding domain (RBD)-specific lateral flow assay (LFA) with a nucleocapsid protein (NCP) IgG ELISA to assess seroconversion in n = 7241 individuals. The majority had experienced symptoms consistent with COVID-19, but had no access to RT-PCR testing. Longitudinal follow-up in n = 97 LFA + individuals was performed up to 20 weeks after initial infection using NCP and spike protein S1 domain (S1) IgG ELISAs and a surrogate virus neutralization test (sVNT).

Results: Individuals reporting symptoms from January 2020 onwards showed seroconversion, as did a considerable proportion of asymptomatic individuals. Seroconversion for symptomatic and asymptomatic individuals was higher in an area with a known infection cluster compared to a low incidence area. Overall, 94% of individuals with a positive IgG result by LFA were confirmed by NCP ELISA. The proportion of ELISA-confirmed LFA results declined over time, in line with contracting NCP IgG titres during longitudinal follow-up. Neutralizing antibody activity was considerably more stable than S1 and NCP IgG titres, and both reach a plateau after approximately 100 d. The sVNT proved to be not only highly specific, but also more sensitive than the specificity-focussed two-tiered serology approach.

Conclusions: Our results demonstrate the high specificity of two-tiered serology testing and highlight the sVNT used as a valuable tool to support modelling of SARS-CoV-2 transmission dynamics, complement molecular testing and provide relevant information to individuals.

Keywords: ELISA; SARS-CoV-2; lateral flow assay; longevity; neutralizing antibodies; serology.

Figure 1.

Overview of immunoassay verification and implementation for SARS-CoV-2 seroconversion screening. Verification of assay specificity and sensitivity was conducted using pre-COVID-19 serum samples and a small set of serum samples from PCR-confirmed COVID-19 cases in March/April 2020 (A). The verified 2-tired strategy for seroconversion screening was then applied from mid-April to mid-August 2020 to test a large cohort of individuals with (mostly symptomatic) suspected prior SARS-VoV-2 infection or exposure (B). A subset of n = 97 individuals was followed up for up to 150 d past the onset of symptoms (C). *The surrogate virus neutralization test was only verified in late May 2020, when it received a CE mark and became commercially available. It was therefore not included as a candidate for the 2-tired testing strategy during the initial verification phase.

Figure 2.

SARS-CoV-2 LFA and NCP IgG ELISA results by area. LFA and NCP ELISA results are shown for n = 7241 individuals that were tested by either Boson or BIOSYNEX LFA. N = 97 individuals that tested positive by LFA did not provide a follow-up serum sample for EUROIMMUN NCP IgG ELISA. Data are shown as the proportion of individuals tested in the indicated areas, stratified depending on whether or not symptoms were reported. Symptoms include common cold symptoms, cough, fever, pneumonia and loss of smell or taste. LFA: lateral flow assay; NCP: nucleocapsid protein.

Figure 3.

Confirmation of BIOSYNEX LFA results by EUROIMMUN NCP IgG ELISA. BIOSYNEX LFA results per month of testing (n = 1003), stratified into individuals with solitary IgM or IgG bands or IgM + IgG + bands (A). IgG levels of anti-SARS-CoV-2 NCP were determined during 2nd tire serological follow-up in n = 935 individuals that were positive by BIOSYNEX LFA. The proportion of samples negative, borderline or positive by NCP IgG ELISA is shown (B) for individuals with solitary IgM or IgG bands, those with both IgM + and IgG + bands and separately for those with a strong solitary IgG band, and for (C) all LFA IgM and/or IgG positive individuals per month of testing. LFA: lateral flow assay; NCP: nucleocapsid protein.

Figure 4.

Stability of anti-NCP, anti-S1 and neutralizing antibodies. (A) The number of individuals who reported onset of symptoms is shown per calendar week. (B) The percent change in IgG levels of anti-SARS-CoV-2 NCP and S1 antibodies as well as the level of inhibition conferred by anti-SARS-CoV-2 RBD-neutralizing antibodies between the two assay time points per donor are plotted against the time between first and second ELISA/sVNT for n = 97 SARS-CoV-2 sero-positive individuals regardless of presence or time of onset of reported symptoms. Data were analysed by simple linear regression. No percent change was calculated for donors whose antibody levels at time point 1 were below the cut-off (n = 5 for NCP, n = 2 for sVNT). (C) Change in anti-NCP or anti-S IgG antibodies and anti-RBD neutralizing antibodies measured by sVNT in a time interval of ≤5 weeks (n = 12) and >6 weeks (n = 85) between first and second ELISA/sVNT. Data were analysed by mixed-effects model analysis with Holm–Sidak’s multiple comparison test. ***p < .0001. (D) Proportion of individuals with declining, increasing or stable antibody levels in the group with >6 weeks (n = 85) between first and second ELISA/sVNT. No percent change was calculated for those donors whose antibody levels at time point 1 were below the cut-off (n = 4 for NCP, n = 1 for sVNT). Changes in antibody levels were calculated by as percent change (test at time point 2 compared to test at time point 1). An increase or decrease of 20% was considered a substantial change. A change of less than ± 20% was considered stable (grey shaded area in C). sVNT: surrogate virus neutraliation test; NCP: nucleocapsid protein; RBD: receptor binding domain.