Antibody Screening Results for Anti-Nucleocapsid Antibodies Towards the Development of a SARS-CoV-2 Nucleocapsid Protein Antigen Detecting Lateral Flow Assay

Abstract

<p>The global COVID-19 pandemic has created an urgent demand for accurate rapid point of care diagnostic tests. Antigen-based assays are suitably inexpensive and can be rapidly mass-produced, but sufficiently accurate performance requires highly-optimized antibodies and assay conditions. An automated liquid handling system, customized to handle lateral flow immunoassay (LFA) arrays, was used for high-throughput antibody screening of anti-nucleocapsid antibodies that will perform optimally on an LFA. Six hundred seventy-three anti-nucleocapsid antibody pairs were tested as both capture and detection reagents with the goal of finding those pairs that have the greatest affinity for unique epitopes of the nucleocapsid protein of SARS-CoV-2 while also performing optimally in an LFA format. In contrast to traditional antibody screening methods (e.g. ELISA, bio-layer interferometry), the methods described here integrate real-time LFA reaction kinetics and binding directly on nitrocellulose. We have identified several candidate antibody pairs that are suitable for further development of an LFA for SARS-CoV-2. </p>

Developing a Paper-Based Antigen Assay to Differentiate between Coronaviruses and SARS-CoV-2 Spike Variants

“…In comparison, others have reported similar performance with a LOD of 0.62 ng/mL (0.0125 nM) for half strips for SARS-CoV-2 nucleocapsid (N) proteins. 20 , 35 We caveat that the LOD measurement was based on purified antigens in solution and not the full virus capsid in biological fluids, and matrix effects in patient samples or differences in spike secondary structure could influence the performance. We estimate the average number of spike proteins in sputum swabs to be in the pM range for most cases and nM range in more severe cases.…”

Developing a Paper-Based Antigen Assay to Differentiate Between Coronaviruses and SARS-CoV-2 Spike Variants

“…In comparison, others have reported similar performance with a LOD of 0.62 ng/ml (0.0125 nM) for half strips for SARS-CoV-2 nucleocapsid (N) proteins. 20,35 We caveat that the LOD measurement was based on purified antigens in solution and not the full virus capsid in biological fluids, and matrix effects in patient samples or differences in spike secondary structure could influence performance. We estimate that the average number of Spike proteins in sputum swabs may be in the pM range for most cases and nM range in more severe cases.…”

“…9 We evaluated the ability of the immunoprobes to form an immunological sandwich in a dipstick assay (Figure 1c). 20 Sandwich formation relies on two reactions, immunoprobeantigen and printed antibody-antigen binding. The choice of both antibodies is primary during test design, where antibody pairs (on the immunoprobe / printed on the paper) with varying affinity and selectivity for a target can be used to detect a wider variety of antigens and develop a binding pattern.…”

Quantifying SARS-Cov-2 Nucleocapsid Antigen in Oropharyngeal Swabs Using Single Molecule Array Technology

“…The SARS-CoV-2 nucleocapsid antigen in-house assay was developed on the automated Simoa HD-1 Analyzer platform (Quanterix©, Billerica, MA, USA). The following antibodies were tested (40143-R001, 40143-R004, 40143-MM05, 40143-MM08 (Sino Biological, Beijing, China) to nd the best performing antibody pair (9). Capture antibodies were covalently attached by standard carbodiimide coupling chemistry to carboxylated paramagnetic beads (Quanterix) using 0.2 mg/mL of antibody and 0.3 mg/mL of EDAC (Thermo Fisher Scienti c, Waltham, MA, USA).…”