Development of a High-Throughput Homogeneous AlphaLISA Drug Screening Assay for the Detection of SARS-CoV-2 Nucleocapsid

Kirill Gorshkov; Catherine Z. Chen; Miao Xu; Juan Carlos De La Torre; Luis Martinez-Sobrido; Thomas Moran; Wei Zheng

doi:10.1021/acsptsci.0c00122

Development of a High-Throughput Homogeneous AlphaLISA Drug Screening Assay for the Detection of SARS-CoV-2 Nucleocapsid

Kirill Gorshkov, Catherine Z. Chen, Miao Xu, Juan Carlos De La Torre, Luis Martinez-Sobrido, Thomas Moran, Wei Zheng

Research output: Contribution to journal › Article › peer-review

7 Scopus citations

Abstract

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is in urgent need of therapeutic options. High-throughput screening (HTS) offers an opportunity to rapidly identify such compounds. In this work, we have developed a homogeneous cell-based HTS system using AlphaLISA detection technology for the SARS-CoV-2 nucleocapsid protein (NP). Our assay measures both recombinant and endogenous NP from viral lysates and tissue culture supernatants (TCS) in a sandwich-based format using two monoclonal antibodies against the NP analyte. Viral NP was detected and quantified in both tissue culture supernatants and cell lysates, with large differences observed between 24 and 48 h of infection. We simulated viral infection by spiking recombinant NP into 384-well plates with live Vero-E6 cells and were able to detect the NP with high sensitivity and a large dynamic range. Antiviral agents that inhibit either viral cell entry or replication decrease the AlphaLISA NP signal. Thus, this assay can be used for high-throughput screening of small molecules and biologics in the fight against the COVID-19 pandemic.

Original language	English
Pages (from-to)	1233-1241
Number of pages	9
Journal	ACS Pharmacology and Translational Science
Volume	3
Issue number	6
DOIs	https://doi.org/10.1021/acsptsci.0c00122
State	Published - 11 Dec 2020

Keywords

AlphaLISA
COVID-19
SARS-CoV-2
high-throughput screening
homogeneous assay
nucleocapsid

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title = "Development of a High-Throughput Homogeneous AlphaLISA Drug Screening Assay for the Detection of SARS-CoV-2 Nucleocapsid",

abstract = "The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is in urgent need of therapeutic options. High-throughput screening (HTS) offers an opportunity to rapidly identify such compounds. In this work, we have developed a homogeneous cell-based HTS system using AlphaLISA detection technology for the SARS-CoV-2 nucleocapsid protein (NP). Our assay measures both recombinant and endogenous NP from viral lysates and tissue culture supernatants (TCS) in a sandwich-based format using two monoclonal antibodies against the NP analyte. Viral NP was detected and quantified in both tissue culture supernatants and cell lysates, with large differences observed between 24 and 48 h of infection. We simulated viral infection by spiking recombinant NP into 384-well plates with live Vero-E6 cells and were able to detect the NP with high sensitivity and a large dynamic range. Antiviral agents that inhibit either viral cell entry or replication decrease the AlphaLISA NP signal. Thus, this assay can be used for high-throughput screening of small molecules and biologics in the fight against the COVID-19 pandemic.",

keywords = "AlphaLISA, COVID-19, SARS-CoV-2, high-throughput screening, homogeneous assay, nucleocapsid",

author = "Kirill Gorshkov and Chen, {Catherine Z.} and Miao Xu and {Carlos De La Torre}, Juan and Luis Martinez-Sobrido and Thomas Moran and Wei Zheng",

note = "Funding Information: This research was supported in part by the Intramural Research Program of the National Center for Advancing Translational Sciences, NIH. We thank Dr. Arturo Gonzalez-Moya and his team at PerkinElmer for assay development support. Publisher Copyright: {\textcopyright} 2020 American Chemical Society. All rights reserved.",

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AU - Chen, Catherine Z.

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AU - Carlos De La Torre, Juan

AU - Martinez-Sobrido, Luis

AU - Moran, Thomas

AU - Zheng, Wei

N1 - Funding Information: This research was supported in part by the Intramural Research Program of the National Center for Advancing Translational Sciences, NIH. We thank Dr. Arturo Gonzalez-Moya and his team at PerkinElmer for assay development support. Publisher Copyright: © 2020 American Chemical Society. All rights reserved.

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N2 - The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is in urgent need of therapeutic options. High-throughput screening (HTS) offers an opportunity to rapidly identify such compounds. In this work, we have developed a homogeneous cell-based HTS system using AlphaLISA detection technology for the SARS-CoV-2 nucleocapsid protein (NP). Our assay measures both recombinant and endogenous NP from viral lysates and tissue culture supernatants (TCS) in a sandwich-based format using two monoclonal antibodies against the NP analyte. Viral NP was detected and quantified in both tissue culture supernatants and cell lysates, with large differences observed between 24 and 48 h of infection. We simulated viral infection by spiking recombinant NP into 384-well plates with live Vero-E6 cells and were able to detect the NP with high sensitivity and a large dynamic range. Antiviral agents that inhibit either viral cell entry or replication decrease the AlphaLISA NP signal. Thus, this assay can be used for high-throughput screening of small molecules and biologics in the fight against the COVID-19 pandemic.

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Development of a High-Throughput Homogeneous AlphaLISA Drug Screening Assay for the Detection of SARS-CoV-2 Nucleocapsid

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