Correlation of SARS-CoV-2 Nucleocapsid Antigen and RNA Concentrations in Nasopharyngeal Samples from Children and Adults Using an Ultrasensitive and Quantitative Antigen Assay

Pollock, Nira R.; Savage, Timothy J.; Wardell, Hanna; Lee, Rose; Mathew, Anu; Stengelin, Martin; Sigal, George B.

doi:10.1128/jcm.03077-20

Cited by 75 publications

(61 citation statements)

References 23 publications

(12 reference statements)

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“…In saliva, also potentially suitable for home collection, we detected N-protein in >90% of COVID-19 PCR+ donors. When analyzing N-protein in longitudinal saliva and DBS samples from an infected donor, we observed that N-protein presented in both saliva and blood before symptom onset and that N-protein levels correlate with Ct values for RNA in saliva, as has been recently observed for N-protein in NP swabs 26 . Recent work suggests that viral load in saliva is a predictor of mortality 27 .…”

Section: Discussionsupporting

confidence: 72%

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

et al. 2021

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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.

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Section: Discussionsupporting

confidence: 72%

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

et al. 2021

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“…The LLD of the S-PLEX SARS-CoV-2 N assay was 0.16 pg/ml (0.16 fg/µl) and a threshold value of 2 X LLD (0.32 pg/ml) was used as a cut-off for classifying samples as positive or negative. It has been previously calculated that one viral RNA copy corresponds to 0.15 fg of N antigen, indicating an extremely high analytical sensitivity for the S-PLEX assay of ∼1–2 RNA copies/µl 11 . This LLD of the S-PLEX assay is on par with the sensitivity of most PCR-based methods and is at least 500 times more sensitive than most commercial LFA-based SARS-CoV-2 antigen assays 11 .…”

Section: Resultsmentioning

confidence: 99%

“…It has been previously calculated that one viral RNA copy corresponds to 0.15 fg of N antigen, indicating an extremely high analytical sensitivity for the S-PLEX assay of ∼1–2 RNA copies/µl 11 . This LLD of the S-PLEX assay is on par with the sensitivity of most PCR-based methods and is at least 500 times more sensitive than most commercial LFA-based SARS-CoV-2 antigen assays 11 . Samples were run in duplicate, where all coefficients of variation (CVs) observed were generally below 10%.…”

Section: Resultsmentioning

confidence: 99%

“…Using a novel electrochemiluminescence (ECL)-based immunoassay (from now on referred to as S-PLEX assay), we demonstrate the ultrasensitive SARS-COV-2 nucleocapsid (N) antigen detection in saliva. The analytical sensitivity at our assay threshold (< 0.32 pg/ml), corresponds to 4 viral RNA copies/µl 11 , on par with the sensitivity of traditional PCR-based molecular tests. Using this quantitative and sensitive technique we also characterized the range of antigen concentrations found in the saliva of COVID-19 patients, generating information that will be critical in establishing the required performance characteristics for future saliva-based rapid antigen tests.…”

Section: Introductionmentioning

confidence: 82%

“…To date, the widespread use of saliva-based SARS-CoV-2 rapid antigen testing has been hampered by the relatively low analytical sensitivity of the lateral flow assays (LFAs) that make up the bulk of the available antigen tests. Typical lower limits of detection (LLD) for SARS-CoV-2 LFAs exceed 100 pg/ml 11 . When applied to saliva, these tests are suitable for detecting cases with high viral loads (e.g.…”

Section: Introductionmentioning

confidence: 99%

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Ultrasensitive assay for saliva-based SARS-CoV-2 antigen detection

Ren

Sohaei

Zacharioudakis

et al. 2021

Preprint

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Widespread SARS-CoV-2 testing is highly valuable for identifying asymptomatic/pre-symptomatic individuals to slow community disease transmission. However, there remains a technological gap for highly reliable, easy, and quick SARS-CoV-2 diagnostic tests that are suitable for frequent mass testing. Compared to the conventional nasopharyngeal (NP) swab-based tests, saliva-based methods are attractive due to easier and safer sampling protocols. Despite its merits in rapid turn-around-time and high throughput compared to traditional PCR-based technologies, the widespread use of saliva-based SARS-CoV-2 rapid antigen tests is hindered by limited analytical sensitivity of current methods. Here, we report the first ultrasensitive, saliva-based SARS-CoV-2 antigen assay with an analytical sensitivity of < 0.32 pg/ml, corresponding to 4 viral RNA copies/ul, which is comparable to that of PCR-based tests. Using the novel electrochemiluminescence (ECL)-based S-PLEX immunoassay, we measured the SARS-CoV-2 nucleocapsid (N) antigen concentration in 105 saliva samples obtained from non-COVID-19 and COVID-19 patients. Our assay displayed absolute specificity and high sensitivity (90.2%), where it correctly identified samples with viral loads up to 35 CT cycles by saliva-based PCR. Paired NP swab-based PCR results were also obtained for 86 cases for comparison. Our assay showed high concordance with saliva-based and NP swab-based PCR in samples with negative (< 0.32 pg/ml) and strongly positive (> 2 pg/ml) N antigen concentrations. Our study unveiled the ultrasensitivity and specificity of the saliva-based S-PLEX assay, demonstrating its clinical value as a high throughput, complementary alternative to PCR-based techniques. The novel technique is especially valuable in cases where compliance to frequent swabbing may be problematic (e.g. schools, nursing homes, etc.).

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