An Optimized High-Throughput Immuno-Plaque Assay for SARS-CoV-2

Alberto A Amarilla; Naphak Modhiran; Yin Xiang Setoh; Nias Y G Peng; Julian D J Sng; Benjamin Liang; Christopher L D McMillan; Morgan E Freney; Stacey T M Cheung; Keith J Chappell; Alexander A Khromykh; Paul R Young; Daniel Watterson

doi:10.3389/fmicb.2021.625136

An Optimized High-Throughput Immuno-Plaque Assay for SARS-CoV-2

Front Microbiol. 2021 Feb 12:12:625136. doi: 10.3389/fmicb.2021.625136. eCollection 2021.

Authors

Alberto A Amarilla¹, Naphak Modhiran^{1

2}, Yin Xiang Setoh¹, Nias Y G Peng¹, Julian D J Sng¹, Benjamin Liang¹, Christopher L D McMillan¹, Morgan E Freney¹, Stacey T M Cheung¹, Keith J Chappell^{1

2

3}, Alexander A Khromykh^{1

3}, Paul R Young^{1

2

3}, Daniel Watterson^{1

2

3}

Affiliations

¹ School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD, Australia.
² The Australian Institute for Biotechnology and Nanotechnology, The University of Queensland, St Lucia, QLD, Australia.
³ Australian Infectious Disease Research Centre, The University of Queensland, St Lucia, QLD, Australia.

Abstract

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 and is capable of human-to-human transmission and rapid global spread. The rapid emergence and global spread of SARS-CoV-2 has encouraged the establishment of a rapid, sensitive, and reliable viral detection and quantification methodology. Here, we present an alternative assay, termed immuno-plaque assay (iPA), which utilizes a combination of plaque assay and immunofluorescence techniques. We have extensively optimized the conditions for SARS-CoV-2 infection and demonstrated the great flexibility of iPA detection using several antibodies and dual-probing with two distinct epitope-specific antibodies. In addition, we showed that iPA could be utilized for ultra-high-throughput viral titration and neutralization assay within 24 h and is amenable to a 384-well format. These advantages will significantly accelerate SARS-CoV-2 research outcomes during this pandemic period.

Keywords: SARS-CoV-2; coronaviruses; immuno-plaque assay (iPA); viral quantification.