The optimization of an ultra-sensitive single molecule assay for the multiplexed detection of SARS-CoV-2 antigens

Abstract

Introduction/Objective: SARS-CoV-2 antigens, including the nucleocapsid (N) protein, spike protein, and its S1 subunit have served as key biomarkers for research and diagnostic purposes. We previously developed quantitative single molecule array (Simoa) assays to measure the concentration of spike, S1 subunit and N protein in plasma samples with femtomolar limits of detection. We aimed to test antibodies that were not available early in the pandemic, reduce assay cross-reactivity, develop a multiplexed assay for spike, S1, and N protein in order to minimize the sample volume needed. Methods/Case Report: Using the Simoa platform, a bead-based digital enzyme-linked immunosorbent assay, we cross-tested 17 S1 subunit and spike antibodies for a total of 130 antibody-pair combinations, we performed dilution linearity experiments to determine the ideal dilution factor, spike and recovery experiments, tested the assay using S1 subunit from other human coronavirus HKV1, NL63, and 229E, pre-pandemic plasma samples from patients that were sick with viral or bacterial respiratory infections. We then used the best antibody pairs to measure S1 and spike in plasma samples collected from patients with severe SARS-CoV-2. Lastly, we conjugated the best-performing capture antibodies for spike, S1 and N to beads labeled with different fluorophores to test if the assay for all three antigens could be multiplexed. Results (if a Case Study enter NA): We observed no cross-reactivity with S1 from other coronavirus strains, no detection of S1 or spike in a cohort of 30 pre-pandemic samples and successfully developed a multiplexed assay for the detection of spike, S1, and N protein, enabling us to use 50% less sample volume. Conclusion(s): Reduction of necessary sample volume is important for studies involving multisystem inflammatory syndrome in children (MIS-C), and possible adverse effects of SARS-CoV-2 vaccinations on children and young adults. An improved assay with minimal cross-reactivity will also be useful to study individuals with post-acute sequelae of SARS-CoV-2 infection (PASC).