A Reagent and Virus Benchmarking Panel for a Uniform Analytical Performance Assessment of N Antigen–Based Diagnostic Tests for COVID-19

Abstract

Rapid diagnostic tests (RDTs) that detect antigen indicative of SARS-CoV-2 infection can help in making quick health care decisions and regularly monitoring groups at risk of infection. With many RDT products entering the market, it is important to rapidly evaluate their relative performance. Comparison of clinical evaluation study results is challenged by protocol design variations and study populations. Laboratory assays were developed to quantify nucleocapsid (N) and spike (S) SARS-CoV-2 antigens. Quantification of the two antigens in nasal eluates confirmed higher abundance of N than S antigen. The median concentration of N antigen was 10 times greater than S per genome equivalent. The N antigen assay was used in combination with quantitative RT-PCR to qualify a panel composed of recombinant antigens, inactivated virus, and clinical specimen pools. This benchmarking panel was applied to evaluate the analytical performance of the SD Biosensor STANDARD Q COVID-19 Ag test, Abbott Panbio COVID-19 Ag Rapid Test, Abbott BinaxNOW COVID-19 Ag test, and the LumiraDx SARS-CoV-2 Ag Test. The four tests displayed different sensitivities toward the different panel members, but all performed best with the clinical specimen pool. The concentration for a 90% probability of detection across the four tests ranged from 21 pg/mL to 102 pg/mL of N antigen in the extracted sample. Benchmarking panels provide a quick way to verify the baseline performance of a diagnostic and enable direct comparison between diagnostic tests.

Antigen concentration, viral load, and test performance for SARS-CoV-2 in multiple specimen types

“…Both the STANDARD Q COVID-19 Ag and LumiraDx SARS-CoV-2 Ag tests had previously been evaluated for their analytical performance against antigen concentration using a clinical specimen pool in a laboratory [ 19 ]. The antigen limits of detection, as defined by antigen concentrations at which tests were expected to be positive 90% of the time, were applied as thresholds in the measured antigen concentration in the ANS specimens and used to categorize the predicted test results as detected or not by each rapid test.…”

“…SARS-CoV-2 N concentration was measured using the Meso Scale Discovery platform (Meso Scale Diagnostics, United States), which uses electrochemiluminescence for detection. The assay details have been described previously [16]. Specimens were shipped to PATH (Seattle, WA, USA) where the antigen concentration assay was conducted.…”

Antigen concentration, viral load, and test performance for SARS-CoV-2 in multiple specimen types

“…Both the STANDARD Q COVID-19 Ag and LumiraDx SARS-CoV-2 Ag tests had previously been evaluated for their analytical performance against antigen concentration using a clinical specimen pool in a laboratory. 16 The antigen limits of detection, as defined by antigen concentrations at which tests were expected to be positive 90% of the time, were applied as thresholds in the measured antigen concentration in the ANS specimens and categorize the predicted test results as detected or not by each rapid test. Clinical performance of the two tests among the close contacts in the study was then predicted based on comparison of these results to the gold standard of NPS RT-PCR positive cases.…”

“…SARS-CoV-2 N concentration was measured using the Meso Scale Discovery platform (Meso Scale Diagnostics, United States), which uses electrochemiluminescence for detection. The assay details have been described previously 16 Positive specimens were run in duplicate and discrepant results of coefficient of variation over 20%, or those outside the assay detection range, were repeated. Samples exceeding the dynamic range of the assay were repeated with increased dilution.…”

Specimen sharing for epidemic preparedness: building a local-to-global virtual biorepository framework

“…Large population-based public health and clinical biorepositories have also been established such as the United Kingdom Biobank (https://www.ukbiobank.ac.uk/), the National Health and Nutrition Examination Survey (https://www.cdc.gov/nchs/nhanes/biospecimens/biospecimens.htm), the National Cancer Institute (https://www.cancer.gov/research/infrastructure) as well as biobank hubs such as the German Biobank Node [13]. The COVID-19 pandemic has led to a proliferation of collections both in existing and COVID-19-centric biorepositories (PATH, https://www.path.org/programs/diagnostics/washington-covid-19-biorepository/ [14][15][16]. Yet, despite the existence of these developments there is still room for additional collections as these do not represent the diverse range of geography, ecozones, and demographics needed to support emerging infectious disease research response efforts.…”