N-protein presents early in blood, dried blood and saliva during asymptomatic and symptomatic SARS-CoV-2 infection

Abstract

The COVID-19 pandemic continues to have an unprecedented impact on societies and economies worldwide. There remains an ongoing need for high-performance SARS-CoV-2 tests which may be broadly deployed for infection monitoring. Here we report a highly sensitive single molecule array (Simoa) immunoassay in development for detection of SARS-CoV-2 nucleocapsid protein (N-protein) in venous and capillary blood and saliva. In all matrices in the studies conducted to date we observe >98% negative percent agreement and >90% positive percent agreement with molecular testing for days 1-7 in symptomatic, asymptomatic, and pre-symptomatic PCR+ individuals. N-protein load decreases as anti-SARS-CoV-2 spike-IgG increases, and N-protein levels correlate with RT-PCR Ct-values in saliva, and between matched saliva and capillary blood samples. This Simoa SARS-CoV-2 N-protein assay effectively detects SARS-CoV-2 infection via measurement of antigen levels in blood or saliva, using non-invasive, swab-independent collection methods, offering potential for at home and point of care sample collection.

Fig. 1. Simoa SARS-CoV-2 N-protein measurements differentiate pre-pandemic from PCR+ donors.

a Shown are pre-pandemic sera and COVID-positive by molecular test (PCR+) plasma binned by day from PCR (BocaBio (triangles), U. Bonn (circles). First timepoint (closed symbol) and subsequent timepoints (open symbol) are shown. Data number is denoted as n = unique donors (total data points). Lines denote median value. b Concurrent decrease of N-protein (red) and increase of anti-SARS-CoV-2 spike IgG (blue). Time series with N-protein peak at day 1 (n = 10). c Time series with N-protein peak after day 1 (n = 3). Data were normalized to max and aligned at peak N-protein. Non-linear regression to the mean is shown in dashed lines. Non-normalized data are shown in Supplementary Fig. 2.

Fig. 2. SARS-CoV-2 N-protein and anti-SARS-CoV-2 IgG in capillary blood (DBS) differentiate COVID PCR− from COVID PCR+ donors.

a NP-protein in DBS. b IgG in DBS. Data are binned by day from PCR result. First timepoint (closed symbol) and subsequent timepoints (open symbol) are shown. Data number is denoted as n = unique donors (total data points). Lines denote median value. c N-protein over three collections. d IgG over three collections. Lines connect individual donors over multiple collections for PCR− (blue symbols), PCR+ with symptoms (red symbols), and PCR+ without symptoms (open red symbols) DBS. PCR+ n = donors without symptoms (total donors); PCR− n = total donors. Donor 12 is highlighted.

Fig. 3. SARS-CoV-2 N-protein and anti-SARS-CoV-2 IgG levels in capillary blood (DBS) correlate with symptom severity indicators.

a SARS-CoV-2 N-protein and b IgG concentrations segregated by symptom severity. First timepoint (closed symbol) and subsequent timepoints (open symbol) are shown. Data number is denoted as n = unique donors (total data points). Lines denote median value. c Ranking of donors with increasing N-protein levels, and color-coding associated with disease severity. Data are grouped by donors (n = 11), with each bar representing a single sample from 1 of the 3 collections (n = 11 sample collection 1; n = 6 sample collection 2; n = 5 sample collection 3). d IgG for same donors/collections as in c.

Fig. 4. SARS-CoV-2 N-protein in saliva differentiate COVID PCR− from PCR+ donors and correlate with capillary blood (DBS).

a N-protein concentration is binned by day from symptom onset. n = individual donors. b Matched saliva and DBS longitudinal samples from two donors. N-protein in saliva (blue line) and DBS (red line) and IgG in DBS (black line) for the index case (closed symbols) and housemate (lines). Open symbols represent days when symptoms are present for the housemate. c N-protein in matched saliva (blue symbols) and DBS (red symbols) as a function of cycle threshold (Ct) values, with exponential fits (solid lines). d Scatter plot of N-protein in saliva vs DBS.