Addressing variability in rapid antigen detection kits with viral protein reference materials

Lateral flow assay (LFA) rapid antigen detection tests (RADT) became a widely used diagnostic tool during the COVID-19 pandemic, and they remain an indispensable strategy to identify infected individuals and prevent transmission of the SARS-CoV-2 virus in vulnerable populations. Many hundreds of these COVID 19 RADTs have been developed worldwide and are commercially available, yet a lack of relevant comparative analyses required to inform on absolute analytical sensitivity and performance has limited end-user ability to accurately compare brands for decision making. Achieving comparability between manufacturers presents a significant metrological challenge yet is indispensable to both effective regulation and consumer confidence.

The analytical sensitivity, or limit of detection, of COVID-19 RADTs is most often reported as the median tissue culture infectious dose (TCID50) of intact viral culture. However, the vast majority of test kits detect only the viral nucleocapsid protein (Np). As viral cultures are notoriously difficult to standardize and can be challenging to ship across international borders during a pandemic, we developed two Np reference materials (RM) – a fluorescent-labelled Np fusion protein (NP-GFP) using bacterial expression, and an unlabeled protein (NCAP-1) produced in mammalian cells. The protein contents of the RMs were assigned using isotope dilution mass spectrometry and/or spectroscopic detection. Protein higher-order structure (HOS) can affect how efficiently LFA detection antibodies recognize their antigen. Protein HOS was interrogated via size-exclusion chromatography, which revealed that NP-GFP existed exclusively as a tetramer, whereas NCAP-1 consisted of a mixture of tetramers and hexamers. We then assessed the analytical sensitivities of 26 commercially available RADTs using a dilution series of both protein RMs. Despite the differences in expression system, oligomeric state, and buffer composition, both RMs generated highly comparable results within each RADT. However, we demonstrate orders of magnitude differences in analytical sensitivities among distinct RADTs, and find little correlation with the TCID50 assay values reported by manufacturers. These results indicate that consumers’ choice of test kit can potentially influence their perceived infection status.

The emergence of another viral pandemic would undoubtedly result in the production and public marketing of a plethora of new antigen detection test kits. Our data highlight the need for, and practicality of, readily available protein RMs to insure batch-to-batch quality and accuracy of RADTs across manufacturers. Development and regulation of the requisite antigen protein RMs thus represents a crucial component in a pandemic preparedness strategy.