Assessment of the Abbott BinaxNOW SARS-CoV-2 rapid antigen test against viral variants of concern

Summary As the emergence of SARS-CoV-2 variants brings the global pandemic to new levels, the performance of current rapid antigen tests against variants of concern and interest (VOC/I) is of significant public health concern. Here, we report assessment of the Abbot BinaxNOW COVID-19 Antigen Self-Test. Using genetically sequenced remnant clinical samples collected from individuals positive for SARS-CoV-2, we assessed the performance of BinaxNOW against the variants that currently pose public health threats. We measured the limit of detection of BinaxNOW against various VOC/I in a blinded manner. BinaxNOW successfully detected the Omicron (B.1.1.529), Mu (B.1.621), Delta (B.1.617.2), Lambda (C.37), Gamma (P.1), Alpha (B.1.1.7), Beta (B.1.351), Eta (B.1.525), and P.2 variants and at low viral concentrations. BinaxNOW also detected the Omicron variant in individual remnant clinical samples. Overall, these data indicate that this inexpensive and simple-to-use, FDA-authorized and broadly distributed rapid test can reliably detect Omicron, Delta, and other VOC/I.

With the high prevalence of rapid diagnostic assays currently available to the public in point-of-care (POC) settings and as at-home over-the-counter (OTC) kits, an obvious question is whether these tests can even reliably detect Delta, Omicron, and other variants of concern/interest (VOC/I). The timing of detection is particularly important for Omicron, as it has been shown to have a shorter incubation period than previous variants, and thus may become transmissible faster after infection (Brandal et al., 2021;Burki, 2021;Jansen, 2021). If these home and community-based tests can indeed detect VOC/I, they can be implemented as part of broad public health strategies to help curtail the rapid spread of VOC/I. On the other hand, if these rapid tests cannot reliably detect the most prevalent VOC/I, their overall clinical utility at the current point of the pandemic should be called into question.
To that end, here, we report our objective assessment of Abbott's BinaxNOW COVID-19 Antigen Self-Test, which has among the highest usage, availability, distribution, and production rates of rapid tests and was the first lateral flow assay (LFA)-based rapid antigen test to receive U.S. FDA Emergency Use Authorization (EUA) for the home OTC setting (Shah et al., 2021a;Prince-Guerra, 2021;Pollock et al., 2021;Hodges, 2021). BinaxNOW is a SARS-CoV-2 diagnostic assay that detects the viral nucleocapsid (N) protein in samples collected by anterior nasal swab and reports a qualitative positive, negative, or invalid result (BINAXNOW COVID-19 AG CARD, n,d). We previously assessed the usability of BinaxNOW  (Frediani et al., 2021). Here, we report with a higher molecular resolution on the performance of BinaxNOW using a comprehensive panel of VOC/I that is currently of the highest public health significance.
In September 2021, the Mu (B.1.621) variant had been detected in every state in the US and was designated as a VOC by the World Health Organization (WHO), though it was later reclassified as a VOI (World Health Organization, 2022). Importantly, the Mu variant harbors a mutation, E484K, that likely enables the virus to blunt vaccine and infection-induced immunity (Wadman, 2021). Given the potential at the time for this variant to significantly worsen the outlook of the global pandemic, we tested the performance of Binax-NOW in duplicate and a blinded manner using remnant clinical samples (RCS) (Figure 1).
Most recently, the Omicron variant (B.1.1.529) has emerged and been designated a VOC by the WHO due to a high number of mutations in the spike protein (World Health Organization, 2021). The Omicron variant has 32 additional mutations in the spike protein, which may allow it to partially evade vaccine-elicited immunity by escaping neutralizing antibodies from previous strains of SARS-CoV-2 (Cao et al., 2021;Cele et al., 2021;Liu et al., 2021). Owing to its increased transmissibility, Omicron has quickly become the dominant variant in the United States and around the world (CDC, 2020). For these reasons, we assessed the performance of BinaxNOW using pooled, heat-inactivated RCS positive for Omicron (B.1.1.529) variant.

RESULTS
Overall, as detailed in Table 1   were also serially diluted and again assessed with RT-qPCR, to assess the limit of detection of the assay.  Figure 2). This value is lower than the highest detected value of other variants of concern, which may indicate that the BinaxNOW has lower sensitivity against the Omicron (B.1.1.529) variant compared to the previous VOC ( Figure 2). BinaxNOW also demonstrated a lower sensitivity against pooled live Omicron (B.

DISCUSSION
Although the sensitivity of BinaxNOW appears to be slightly decreased against the Omicron (B.1.1.529) variant compared to previous VOC/I, the results still justify the continued use of this readily available, inexpensive, and simple-to-use rapid test kit as part of the community-and/or home-based testing strategies to combat the ongoing public health crisis. Ultimately, laboratory experiments cannot fully recapitulate the real-world application of a test kit, and the utility of the BinaxNOW will depend on a review of its clinical performance, which we are currently conducting.

Limitations of the study
We acknowledge the limitations of the current study are that we used RCS that may have some degradation and may not accurately reflect real-world testing conditions. In addition, the BinaxNOW kit lots used in this study varied from experiment to experiment and that could have generated slight differences in the test performance. Additional studies into the quantitative differences in the N antigen levels of Omicron variant patient samples will help to clarify the implications of the decreased performance. Future studies are also underway to compare BinaxNOW to other available rapid antigen tests.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
Abbott BinaxNOW COVID-19 Antigen Self-Test was graciously provided by the U.S. Department of Health and Human Services and Bruce J. Tromberg of the National Institute of Biomedical Imaging and iScience Article obtained from individuals (N = 8) infected with the B.1.2 strain of SARS-CoV-2, which is not considered to be a VOC/I by the WHO, and therefore served as our comparator. RT-qPCR for the SARS-CoV-2 N2 gene using CDC primers/probe set was performed on each RCS and N2 Ct values were used as estimates of viral load.
In addition, using genetically sequenced RCS collected from individuals positive for SARS-CoV-2 across the country, we created RCS ''pools'' of each of the VOC/I (Figure 1). The individual VOC/I pools were verified by repeating genetic sequencing to ensure quality control. Panels of the VOC/I pools of varying viral loads were then created by serial dilution using SARS-CoV-2 negative pooled human donor nasal wash (Lee Biosolutions, Catalog No. 991-26-P-1). Dilutions of every pool were then analyzed by RT-qPCR for the SARS-CoV-2 N2 gene using CDC primers/probe set (Figure 1). The performance of BinaxNOW was then assessed in a blinded manner in triplicate.
The pools for heat-inactivated Omicron variant were created using the same method as previously stated ( Figure 1) and diluted to 10 dilutions that ranged from a Ct value of 21.2 to a Ct value of 31.7. The limit of detection was determined by testing each sample dilution 5 times with BinaxNOW. For testing, we used the direct spike method, where 20mL of sample was spiked onto the swab provided with the test and subsequent steps were according to the BinaxNOW instructions for use (IFU). This limit of detection was further confirmed by testing the highest detectable dilution as well as the two neighboring dilutions a further 20 times.
We then assessed BinaxNOW using non heat inactivated RCS obtained from the University of Washington that were confirmed to be Omicron (B.1.1.529). These samples were pooled and diluted to a range of Ct values between 19.3 and 28.8. The dilutions were tested according to the IFU of BinaxNOW rapid antigen test to determine the limit of detection. Finally, we obtained 12 RCS sequence confirmed to be Omicron from LabCorp and measured N2 Ct in triplicate by RT-qPCR (Table 2). These samples were tested in duplicate using BinaxNOW, following the IFU.

QUANTIFICATION AND STATISTICAL ANALYSIS
The mean and standard deviation of the Ct values in Tables 1 and 2