Scalable, Micro-Neutralization Assay for Assessment of SARS-CoV-2 (COVID-19) Virus-Neutralizing Antibodies in Human Clinical Samples
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Abstract
As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic expanded, it was clear that effective testing for the presence of neutralizing antibodies in the blood of convalescent patients would be critical for development of plasma-based therapeutic approaches. To address the need for a high-quality neutralization assay against SARS-CoV-2, a previously established fluorescence reduction neutralization assay (FRNA) against Middle East respiratory syndrome coronavirus (MERS-CoV) was modified and optimized. The SARS-CoV-2 FRNA provides a quantitative assessment of a large number of infected cells through use of a high-content imaging system. Because of this approach, and the fact that it does not involve subjective interpretation, this assay is more efficient and more accurate than other neutralization assays. In addition, the ability to set robust acceptance criteria for individual plates and specific test wells provided further rigor to this assay. Such agile adaptability avails use with multiple virus variants. By February 2021, the SARS-CoV-2 FRNA had been used to screen over 5000 samples, including acute and convalescent plasma or serum samples and therapeutic antibody treatments, for SARS-CoV-2 neutralizing titers.
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SciScore for 10.1101/2021.03.05.434152: (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
Antibodies Sentences Resources Virus and antibody-free cell culture medium served as a negative control. antibody-free cell culture mediumsuggested: NoneThe diluted virus and diluted test samples were mixed 1:1 (vol/vol) and incubated at 37°C in a humidified 5% CO2 atmosphere for 1 h to allow anti-SARS-CoV-2 antibodies to bind the virus. anti-SARS-CoV-2suggested: NoneCells were stained with an anti-SARS antibody (SARS-CoV/SARS-CoV-2 nucleocapsid antibody, rabbit monoclonal antibody; Sino … SciScore for 10.1101/2021.03.05.434152: (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
Antibodies Sentences Resources Virus and antibody-free cell culture medium served as a negative control. antibody-free cell culture mediumsuggested: NoneThe diluted virus and diluted test samples were mixed 1:1 (vol/vol) and incubated at 37°C in a humidified 5% CO2 atmosphere for 1 h to allow anti-SARS-CoV-2 antibodies to bind the virus. anti-SARS-CoV-2suggested: NoneCells were stained with an anti-SARS antibody (SARS-CoV/SARS-CoV-2 nucleocapsid antibody, rabbit monoclonal antibody; Sino Biological #40143-R001), diluted to 0.125 μg/mL in 3% BSA/PBS blocking solution for 1 h at room temperature. anti-SARSsuggested: NoneSARS-CoV/SARS-CoV-2 nucleocapsid antibody,suggested: (Thermo Fisher Scientific Cat# MA5-35943, RRID:AB_2866555)The cells were again washed three times with PBS and then stained with an Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific #A11037), diluted in BSA/PBS for 1 h in the dark at room temperature, and counterstained with Hoechst 33342 nuclear stain (Thermo Fisher Scientific #H3570). anti-rabbit IgGsuggested: (Thermo Fisher Scientific Cat# A-11037, RRID:AB_2534095)In the case of positive samples, S1-specific antibodies will bind to the viral antigens. S1-specificsuggested: NoneTo detect the bound antibodies, a second incubation is carried out using an enzyme-labelled antihuman IgG (peroxidase conjugate) catalyzing a color reaction. antihuman IgGsuggested: (GeneTex Cat# GTX28798, RRID:AB_374523)Following a wash to remove any unbound enzyme-linked antibody, a substrate is added to the wells and color develops in proportion to the amount of IgG antibodies in the sample bound to the SARS-CoV-2 RBD antigen. SARS-CoV-2 RBD antigen.suggested: NoneExperimental Models: Cell Lines Sentences Resources The virus was propagated at the Integrated Research Facility-Frederick in high containment (biosafety level 3 [BSL-3]) by inoculating Vero cells, acquired from the American Type Culture Collection (ATCC #CCL-81; Manassas, VA, USA). Verosuggested: ATCC Cat# CCL-81, RRID:CVCL_0059)#655948) containing Vero E6 cells and incubated at 37°C and 5% CO2 for 24 h. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources The infected cells were incubated for 72 h in Dulbecco’s Modified Eagle Medium with L-glutamine (DMEM; Lonza, Walkersville, MD, USA) containing 2% heat-inactivated fetal bovine serum (FBS; SAFC Biosciences, Lenexa, KS, USA) in a humidified atmosphere at 37°C with 5% carbon dioxide (CO2). SAFCsuggested: (SAFC, RRID:SCR_008554)A four-parameter logistical analysis was performed on the full dilution series using Prism (GraphPad Software, San Diego, CA). Prismsuggested: (PRISM, RRID:SCR_005375)GraphPadsuggested: (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: We found the following clinical trial numbers in your paper:
Identifier Status Title NCT04344977 Completed Collection of Anti-SARS-CoV-2 Immune Plasma NCT04546581 Active, not recruiting Inpatient Treatment With Anti-Coronavirus Immunoglobulin (IT… 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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