Performance of Three SARS-CoV-2 Immunoassays, Three Rapid Lateral Flow Tests, and a Novel Bead-Based Affinity Surrogate Test for the Detection of SARS-CoV-2 Antibodies in Human Serum

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1. Version published to 10.1128/jcm.00319-21

   Jul 19, 2021
2. 

   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2021.02.07.21251062: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Institutional Review Board Statement	Consent: Inclusion criteria were a PCR-confirmed COVID-19 disease with a first positive SARS-CoV-2-PCR at least 14 days before serum collection, age of at least 18 years and a written informed consent. IRB: Use of this samples was approved by the local ethics committee.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	The date of symptom onset, clinical classification and basic demographic information (male/female, age) were recorded.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   In order to neutralize residual input virus, inoculated cells were washed twice with PBS two …

   SciScore for 10.1101/2021.02.07.21251062: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Institutional Review Board Statement	Consent: Inclusion criteria were a PCR-confirmed COVID-19 disease with a first positive SARS-CoV-2-PCR at least 14 days before serum collection, age of at least 18 years and a written informed consent. IRB: Use of this samples was approved by the local ethics committee.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	The date of symptom onset, clinical classification and basic demographic information (male/female, age) were recorded.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   In order to neutralize residual input virus, inoculated cells were washed twice with PBS two hours post infection and new medium containing 1:1000 anti-VSV-G antibody (8G5F11, Kerafast) was added.	anti-VSV-G suggested: (Absolute Antibody Cat# Ab01401-2.0, RRID:AB_2883992)
   The thresholds in the SERION assay were < 10 U/ml for negative results, ≥ 10 U/ml but < 14 U/ml for IgA and ≥ 10 U/ml but < 15 U/ml for IgG antibodies as borderline and > 14 U/ml for IgA and > 15 U/ml for IgG as positive.	IgG suggested: None
   Bead-based surrogate test for the detection of neutralizing anti-SARS-CoV-2 antibodies: Dynabeads®	anti-SARS-CoV-2 suggested: None
   After extensive washing, bead-bound antibodies were detected with Goat-anti-human IgG Alexa 647 (Dianova, Hamburg, Germany)	Goat-anti-human IgG suggested: None
   A mouse/human chimeric monoclonal anti-SARS-CoV2-Spike-S1 antibody (Sino Biological, Beijing, China) was used as a positive control for all measurements.	anti-SARS-CoV2-Spike-S1 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Briefly, VSVΔG-eGFP/luciferase was pseudotyped on BHK cells inducibly expressing the VSV G protein (kindly provided by Gert Zimmer) to generate VSVΔG-G.	BHK suggested: RRID:CVCL_HA32)
   Next, 293T cells were transfected with a plasmid expressing SARS-CoV2 Spike (kindly provided by Markus Hoffmann and Stefan Pöhlmann, (5)) using TransIT-X2 (Mirus) according to the manufacturer’s instructions.	293T suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
   Viral titers were determined on Vero E6 cells by the quantification of green fluorescence.	Vero E6 suggested: RRID:CVCL_XD71)
   Serum dilutions were mixed 1:1 with pseudoparticles and incubated for 45 minutes at 37°C prior to addition to the preplated Vero cells and incubation at 37 °C for 16 hours.	Vero suggested: None
   M-270 Epoxy (ThermoFisher, Waltham, USA) were coated according to the manufacturer’s instructions with different concentrations of either commercially available full-length SARS-CoV2 S1-Spike-Protein (His-tagged, Sino Biological, Beijing, China) expressed and purified from HEK cells or with SARS-CoV-2 RBD protein expressed and purified from Expi293F™ cells (22-26).	HEK suggested: None
   Software and Algorithms
   Sentences	Resources
   Rapid lateral flow tests: The serum samples were also used to evaluate three rapid lateral flow antibody tests: NADAL COVID-19 IgG/IgM Test (whole blood, serum or plasma) from new art laboratories (nal)-von minden (Moers, Germany) targeting a not further specified SARS-CoV-2-Antigen, Panbio COVID-19 IgG/IgM rapid test device from Abbott (Chicago IL, USA) utilizing the nucleocapsid (N) protein (14) and the Cleartest Corona 2019-nCOV IgM/IgG (Servoprax, Wesel, Germany) targeting a “2019-nCOV-Antigen”.	Abbott suggested: (Abbott, RRID:SCR_010477)
   Samples were measured on a FACS Calibur flow cytometer (Becton Dickinson, Franklin Lakes NJ, USA) and FlowJo software version 10 (TreeStar, Ashland OR, USA) was used for data analysis.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Statistical analysis: Data analysis was performed using Microsoft Excel 2016, IBM SPSS Statistics 26, and GraphPad Prism 8.	Microsoft Excel suggested: (Microsoft Excel, RRID:SCR_016137) SPSS suggested: (SPSS, RRID:SCR_002865) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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   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).

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   Limitations of the study are as follows: The severity of disease was not queried and one participant suffered from an immunodeficiency. Recruiting participants in a health care setting resulted in a gender imbalance towards the female gender. Though there are inter-assay differences in sensitivity, the number of participants was not high enough to assess a significant superiority. It is noteworthy that the three immune assays incorporated in this study are based on different antigen components. Whereas the Euroimmun assay is based on S1 antigen, Mikrogen recomWell is based on the nucleocapsid protein as antigen and SERION ELISA agile is coated with both S1 and S2 and the nucleocapsid. In the patient, followed over time IgG dynamics differ between the different assays. Data from participants excluded from the main analysis whose COVID-19 disease was not confirmed by PCR, but by an unknown antibody assay, and who did not show neutralizing antibodies highlights the danger of COVID-19 diagnoses by antibody detection with apparently insufficient assays. In this study only one assay (SERION ELISA agile SARS-CoV-2 IgG) showed a sensitivity and specificity of greater than 98 % and may be suitable for widespread assessment whether sera have neutralizing activity against SARS-CoV-2, for the clarification of doubtful PCR results and to determine if patients with potential post-COVID-19-symptoms have had an undetected COVID-19 disease as well as for epidemiological investigations.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   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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3. 

   Version published to 10.1101/2021.02.07.21251062v1 on medRxiv

   Feb 8, 2021