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

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

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.