A SARS-CoV-2 coronavirus nucleocapsid protein antigen-detecting lateral flow assay

Abstract

Inexpensive, simple, rapid diagnostics are necessary for efficient detection, treatment, and mitigation of COVID-19. Assays for SARS-CoV2 using reverse transcription polymerase chain reaction (RT-PCR) offer good sensitivity and excellent specificity, but are expensive, slowed by transport to centralized testing laboratories, and often unavailable. Antigen-based assays are inexpensive and can be rapidly mass-produced and deployed at point-of-care, with lateral flow assays (LFAs) being the most common format. While various manufacturers have produced commercially available SARS-Cov2 antigen LFAs, access to validated tests remains difficult or cost prohibitive in low-and middle-income countries. Herein, we present a visually read open-access LFA (OA-LFA) using commercially-available antibodies and materials for the detection of SARS-CoV-2. The LFA yielded a Limit of Detection (LOD) of 4 TCID50/swab of gamma irradiated SARS-CoV-2 virus, meeting the acceptable analytical sensitivity outlined by in World Health Organization target product profile. The open-source architecture presented in this manuscript provides a template for manufacturers around the globe to rapidly design a SARS-CoV2 antigen test.

Fig 1. An LFA was constructed using 14 mm of a sample pad (Ahlstrom 8964), 29 mm of a conjugate pad (Lydall 9818), 25 mm of a nitrocellulose analytical membrane (CN95), and 22 mm of wicking pad (Ahlstrom 320).

Intersecting circles indicate the overlap between two materials. The test line and control line are striped at 8 and 14 mm from the downstream end of the nitrocellulose membrane.

Fig 2. Measurable SARS-CoV-2 nucleocapsid released from three different swab types.

SRS with or without SARS-CoV-2 was pipetted onto each swab before being placed in elution buffer. Eluted nucleocapsid protein was measured using the Meso Scale Discovery platform.

Fig 3. Analytical performance of OA-LFA.

(A) Dose-response curve of gamma-irradiated virus spiked into SRS. Concentrations refer to the total amount of genomic material applied to the swab. (B) Validation of LOD using ten negative replicates and ten replicates spiked with SARS-CoV-2 at the LOD concentration.

Fig 4. Comparison of diluent volume.

Swab was placed into a tube containing either 275 or 500 μL of sample diluent and run using the conditions described previously. Each condition was tested with a negative control and irradiated virus diluted in simulated respiratory secretion, run with an n = 3. Reducing the dilution factor increases the signal near our LOD, suggesting an improvement in overall performance.