Development of a high-throughput homogeneous AlphaLISA drug screening assay for the detection of SARS-CoV-2 Nucleocapsid
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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 the research field 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 NP 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 hours and 48 hours of infection. We simulated the viral infection by spiking in 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. Anti-viral agents that inhibit either viral cell entry or replication will 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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SciScore for 10.1101/2020.08.20.258129: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Reagents and Materials: The following item was purchased from ATCC: Vero-E6 (CRL-1586, RRID:CVCL_0574). Vero-E6detected: (IZSLER Cat# BS CL 87, RRID:CVCL_0574)Vero-E6 cell culture: Vero-E6 (grown in EMEM, 10% FBS, and 1% Penicillin/Streptomycin), were cultured in T175 flasks and passaged at 95% confluency. Vero-E6suggested: NoneVero E6 cells were plated at 20,000 and 50,000 cells/well in 384-well plate (TC) and incubated overnight. Vero E6SciScore for 10.1101/2020.08.20.258129: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Reagents and Materials: The following item was purchased from ATCC: Vero-E6 (CRL-1586, RRID:CVCL_0574). Vero-E6detected: (IZSLER Cat# BS CL 87, RRID:CVCL_0574)Vero-E6 cell culture: Vero-E6 (grown in EMEM, 10% FBS, and 1% Penicillin/Streptomycin), were cultured in T175 flasks and passaged at 95% confluency. Vero-E6suggested: NoneVero E6 cells were plated at 20,000 and 50,000 cells/well in 384-well plate (TC) and incubated overnight. Vero E6suggested: RRID:CVCL_XD71)Experimental Models: Organisms/Strains Sentences Resources Vero-E6 cell culture: Vero-E6 (grown in EMEM, 10% FBS, and 1% Penicillin/Streptomycin), were cultured in T175 flasks and passaged at 95% confluency. Vero-E6suggested: NoneSoftware and Algorithms Sentences Resources Statistical analysis and illustrations: Concentration-response curves were fit using non-linear regression, standard curve interpolation, and graphs were generated in Graphpad Prism V8.43. Graphpad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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