SARS-CoV-2 Coronavirus Nucleocapsid Antigen-Detecting Half-Strip Lateral Flow Assay Toward the Development of Point of Care Tests Using Commercially Available Reagents

Abstract

The SARS-CoV-2 pandemic has created an unprecedented need for rapid diagnostic testing to enable the efficient treatment and mitigation of COVID-19. The primary diagnostic tool currently employed is reverse transcription polymerase chain reaction (RT-PCR), which can have good sensitivity and excellent specificity. Unfortunately, implementation costs and logistical problems with reagents during the global SARS-CoV-2 pandemic have hindered its universal on demand adoption. Lateral flow assays (LFAs) represent a class of diagnostic that, if sufficiently clinically sensitive, may fill many of the gaps in the current RT-PCR testing regime, especially in low- and middle-income countries (LMICs). To date, many serology LFAs have been developed, though none meet the performance requirements necessary for diagnostic use cases, primarily due to the relatively long delay between infection and seroconversion. However, on the basis of previously reported results from SARS-CoV-1, antigen-based SARS-CoV-2 assays may have significantly better clinical sensitivity than serology assays. To date, only a very small number of antigen-detecting LFAs have been developed. Development of a half-strip LFA is a useful first step in the development of any LFA format. In this work, we present a half-strip LFA using commercially available antibodies for the detection of SARS-CoV-2. We have tested this LFA in buffer and measured an LOD of 0.65 ng/mL (95% CI of 0.53 to 0.77 ng/mL) ng/mL with recombinant antigen using an optical reader with sensitivity equivalent to a visual read. Further development, including evaluating the appropriate sample matrix, will be required for this assay approach to be made useful in a point of care setting, though this half-strip LFA may serve as a useful starting point for others developing similar tests.

Serum SARS-COV-2 Nucleocapsid Protein: A Sensitivity and Specificity Early Diagnostic Marker for SARS-COV-2 Infection

“…Further studies are needed to confirm whether infected patients have a higher incidence of viremia in the early stages or whether the over-expressed N protein of the lung virus spills into the blood. Recently, many new detection technologies for SARS-COV-2 have been reported, such as luciferase IP system assay and lateral flow assays (Grant et al, 2020;Haljasmägi et al, 2020). Serum SARS-COV-2 antibody test has been widely recognized as serological evidence for the diagnosis of COVID-19 (Cai et al, 2020;Shen et al, 2020;Sun et al, 2020).…”

A serological aptamer-assisted proximity ligation assay for COVID-19 diagnosis and seeking neutralizing aptamers

“…This performance is comparable to that of the commercial ELISA kit with an LOD of approximately 50 pg mL À1 , and much better than that of a recent half-strip lateral ow assay with an LOD of 0.65 ng mL À1 . 44 To demonstrate the selectivity of the aptamer-assisted proximity ligation assay, we performed the PLA-qPCR using different competing proteins and the real-time uorescence intensity of qPCR was recorded (Fig. 2C), including the N protein of SARS-CoV, spike S1, cardiac troponin I (cTnI), interferon-gamma (IFN-g), and bovine serum albumin (BSA).…”

SARS-CoV-2 pandemic: a review of molecular diagnostic tools including sample collection and commercial response with associated advantages and limitations

“…Low-cost, rapid antigenbased tests [106,[114][115][116][117][118]121] POC platforms utilizing isothermal and RT-PCR tests [14,207] CRISPR-based lateral flow assay strips [43] Interpandemic Pandemic phase Interpandemic…”