Ultrasensitive high-resolution profiling of early seroconversion in patients with COVID-19

Abstract

Sensitive assays are essential for the accurate identification of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we report a multiplexed assay for the fluorescence-based detection of seroconversion in infected individuals from less than 1 µl of blood, and as early as the day of the first positive nucleic acid test after symptom onset. The assay uses dye-encoded antigen-coated beads to quantify the levels of immunoglobulin G (IgG), IgM and IgA antibodies against four SARS-CoV-2 antigens. A logistic regression model trained using samples collected during the pandemic and samples collected from healthy individuals and patients with respiratory infections before the first outbreak of coronavirus disease 2019 (COVID-19) was 99% accurate in the detection of seroconversion in a blinded validation cohort of samples collected before the pandemic and from patients with COVID-19 five or more days after a positive nasopharyngeal test by PCR with reverse transcription. The high-throughput serological profiling of patients with COVID-19 allows for the interrogation of interactions between antibody isotypes and viral proteins, and should help us to understand the heterogeneity of clinical presentations.

Fig. 1. Schematic of the Simoa serological assay.

Plasma is incubated with four types of dye-encoded beads that are each coupled to one of four viral targets (spike, S1, RBD and nucleocapsid). IgG, IgA and IgM antibodies specific to the SARS-CoV-2 targets bind to the viral antigen-coated beads. After washing, beads are introduced to biotinylated anti-human immunoglobulin antibodies to label either IgG, IgM or IgA in the different reactions. After additional washes, the enzyme SβG is introduced. The beads are washed, resuspended in fluorogenic RGP and loaded into a 216,000-microwell array for multicolour imaging.

Fig. 2. Profiling seroconversion in COVID-19.

Simoa serological assay results for IgG, IgM and IgA against the four viral targets (spike, S1 subunit, RBD and nucleocapsid) for pre-pandemic samples (light blue; n = 200), nasopharyngeal (NP) PCR-negative samples (dark blue; n = 100) and SARS-CoV-2-positive samples (black (immunocompetent; n = 141) or red (immunosuppressed; n = 31)). The SARS-CoV-2-positive samples were divided into four groups according to time since since the first positive nasopharyngeal RT–PCR test. Black lines indicate the median normalized AEB value of each population.

Fig. 3. Profiling the seroconversion time course in COVID-19.

a–d, Normalized AEB over the 10 d since a positive nasopharyngeal RT–PCR for patients 1 (a), 2 (b), 3 (c) and 4 (d). Patient 1 was a 67-year-old man who recovered 10 d after diagnosis with COVID-19. Patient 2 was a 50-year-old man with multiple comorbidities who died from acute respiratory distress syndrome 20 d after diagnosis with COVID-19. He received remdesivir from days 1–5. Patient 3 was a 50-year-old man with pancytopenia and B-cell acute lymphoblastic leukaemia. He died from acute respiratory distress syndrome 8 d after diagnosis with COVID-19. Patient 4 was an 89-year-old man who died from hypoxaemic respiratory failure 8 d after diagnosis with COVID-19. He received hydroxychloroquine from days 1–5. The circle, square, triangle and star represent the mean of two replicate measurements, while the error bars represent the standard deviation.

Fig. 4. Classification of COVID-19 using Simoa serological assays.

a, Left: ROC curves for all positive cases (n = 141) in the pre-pandemic cohort (n = 199) using either the full marker panel model (green), the early-stage model (blue) or the late-stage model (red). Middle: ROC curves for late-stage cases (more than 1 week after positive nasopharyngeal PCR test; n = 50) in the pre-pandemic cohort (n = 199) using either the full marker panel model (green) or the late-stage model (red). Right: ROC curves for early-stage cases (first week after positive nasopharyngeal PCR test; n = 91) in the pre-pandemic cohort (n = 199) using either the full marker panel model (green) or the early-stage model (blue). AUC values and 95% confidence intervals (CIs) are shown for each graph. b, Simoa serological assay results for IgG, IgM and IgA against the four viral targets (spike, S1 subunit, RBD and nucleocapsid) for pre-pandemic samples (light blue; n = 232) and nasopharyngeal RT–PCR-positive samples (grey; n = 68). Statistical significance was determined using Mann–Whitney U-tests. All U-tests were two tailed and did not correct for multiple comparisons. Black lines indicate the median normalized AEB value of each population.