Quantifying SARS-CoV-2 nucleocapsid antigen in oropharyngeal swabs using single molecule array technology.

Area of Science:

• Biotechnology
• Infectious Diseases
• Molecular Diagnostics

Background:

• Real-time RT-PCR is the standard for SARS-CoV-2 detection but requires specialized equipment.
• Nucleocapsid antigen assays offer potential for rapid detection but require high sensitivity.
• Single Molecule Array (Simoa) technology enables ultra-sensitive biomarker detection.

Purpose of the Study:

• To develop and evaluate a highly sensitive SARS-CoV-2 nucleocapsid antigen assay using Simoa technology.
• To compare the technical and clinical sensitivity of Simoa-based assays with real-time RT-PCR.
• To assess the utility of Simoa nucleocapsid antigen assays for clinical diagnosis and as reference methods.

Main Methods:

• Development of in-house and evaluation of a pre-commercial SARS-CoV-2 nucleocapsid antigen assay on Simoa.
• Testing of 148 SARS-CoV-2 RT-PCR positive and 73 negative oropharyngeal swabs.
• Determination of technical sensitivity using SARS-CoV-2 virus culture material.

Main Results:

• Both Simoa nucleocapsid antigen assays demonstrated high technical sensitivity (approx. 100 RNA molecules/mL).
• Pre-commercial assay: 96% sensitivity, 100% specificity. In-house assay: 95% sensitivity, 100% specificity.
• Assays showed strong correlation (r=0.91) and comparable clinical sensitivity to RT-PCR.

Conclusions:

• Simoa-based SARS-CoV-2 nucleocapsid antigen assays offer high sensitivity and specificity.
• These assays are comparable to RT-PCR for clinical diagnosis of SARS-CoV-2 infection.
• Simoa assays can serve as valuable clinical tools and reference methods for antigen Point of Care Testing.