Abstract
This study aimed to develop a highly sensitive SARS-CoV-2 nucleocapsid antigen assay using the single molecule array (Simoa) technology and compare it with real time RT-PCR as used in routine clinical practice with the ambition to achieve a comparative technical and clinical sensitivity. Samples were available from 148 SARS-CoV-2 real time RT-PCR positive and 73 SARS-CoV-2 real time RT-PCR negative oropharyngeal swabs. For determination of technical sensitivity SARS-CoV-2 virus culture material was used. The samples were treated with lysis buffer and analyzed using both an in-house and a pre-commercial SARS-CoV-2 nucleocapsid antigen assay on Simoa. Both nucleocapsid antigen assays have a technical sensitivity corresponding to around 100 SARS-CoV-2 RNA molecules/mL. Using a cut-off at 0.1 pg/mL the pre-commercial SARS-CoV-2 nucleocapsid antigen assay had a sensitivity of 96% (95% CI 91.4–98.5%) and specificity of 100% (95% CI 95.1–100%). In comparison the in-house nucleocapsid antigen assay had sensitivity of 95% (95% CI 89.3–98.1%) and a specificity of 100% (95% CI 95.1–100%) using a cut-off at 0.01 pg/mL. The two SARS-CoV-2 nucleocapsid antigen assays correlated with r = 0.91 (P < 0.0001). The in-house and the pre-commercial SARS-CoV-2 nucleocapsid antigen assay demonstrated technical and clinical sensitivity comparable to real-time RT-PCR methods for identifying SARS-CoV-2 infected patients and thus can be used clinically as well as serve as a reference method for antigen Point of Care Testing.
Highlights
The assay was compared with a pre-commercial SARS-CoV-2 nucleocapsid antigen assay from Quanterix, but FDA EUA approved on January 11, 2021
We demonstrated that the in-house assay and the Quanterix assay are highly correlated and can measure the nucleocapsid protein with similar technical sensitivity as determined by comparing to a virus culture with a measured concentration of SARS-CoV-2 RNA molecules
The two antigen assays performed equal well and both assays are capable of discriminating real time RT-polymerase chain reaction (PCR) positive samples from real time RT-PCR negative patients with sensitivities of 95–96% and specificities of 96–100% depending on the applied cut-off value
Summary
We determined whether a SARS-CoV-2 antigen assay using our established and highly sensitive technology, the single molecule array (Simoa) system (Quanterix©, Lexington, MA, USA) could serve as an alternative to the conventional PCR test to detect SARS-CoV-2. This would require a clinical sensitivity for diagnostic purposes comparable to the PCR methodology. This paper provides a head-to-head evaluation of both of these antigen-based tests compared to the conventional PCR test serving as the reference test to diagnose SARS-CoV-2
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