A Highly Sensitive and Specific SARS-CoV-2 Spike- and Nucleoprotein-Based Fluorescent Multiplex Immunoassay (FMIA) to Measure IgG, IgA, and IgM Class Antibodies

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1. Version published to 10.1128/spectrum.01131-21

   Dec 22, 2021
2. 

   ScreenIT

   Aug 4, 2021

   SciScore for 10.1101/2021.07.28.21260990: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: Written informed consent was obtained from all volunteers.
   Sex as a biological variable	not detected.
   Randomization	The vaccinee cohort (n=20) was randomly selected from a larger cohort (n=180) of vaccinated healthcare personnel of Turku University Hospital [16] approved by the Southwest Finland health district ethical permission ETMK 19/1801/2020.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Affinipure Goat Anti-Human IgG, IgA or IgM Fcγ Fragment Specific detection antibodies (Jackson Immuno Research) to the wells.	Anti-Human IgG suggested: None
   We measured IgG, IgA, IgM and total Ig antibodies against …

   SciScore for 10.1101/2021.07.28.21260990: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: Written informed consent was obtained from all volunteers.
   Sex as a biological variable	not detected.
   Randomization	The vaccinee cohort (n=20) was randomly selected from a larger cohort (n=180) of vaccinated healthcare personnel of Turku University Hospital [16] approved by the Southwest Finland health district ethical permission ETMK 19/1801/2020.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Affinipure Goat Anti-Human IgG, IgA or IgM Fcγ Fragment Specific detection antibodies (Jackson Immuno Research) to the wells.	Anti-Human IgG suggested: None
   We measured IgG, IgA, IgM and total Ig antibodies against SARS-CoV-2 S1 and N proteins from sera diluted 1:300.	IgG suggested: None IgA suggested: None IgM suggested: None total Ig antibodies against SARS-CoV-2 S1 and N suggested: None SARS-CoV-2 suggested: None S1 suggested: None
   SARS-CoV-2 S1 (GenBank ID: MN908947.3) and N (Genbank ID: NC_045512.2) antigens were expressed and purified, and antigen-specific antibody levels were measured with a Victor Nivo device (PerkinElmer) as described previously [16].	antigen-specific suggested: None
   Comparison of FMIA, EIA and MNT assays: We calculated Spearman correlation coefficient (ρ) and the statistical significance of the correlation between FMIA and EIA antibody concentrations and the two laboratories’ NAb titers.	EIA suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Briefly, the MNT of laboratory #1 was performed using Vero E6 cells and true duplicates of sera diluted serially from 1:4.	Vero E6 suggested: RRID:CVCL_XD71)
   MNT of laboratory #2 used VeroE6-TMPRSS2-H10 cells fixed with 4% formaldehyde and measured CPE after three days.	VeroE6-TMPRSS2-H10 suggested: None
   Software and Algorithms
   Sentences	Resources
   MFI values were converted into FMIA U/ml by interpolation from the 5-PL reference curve with GraphPad Prism v9.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   We determined the thresholds for positivity by comparing all possible threshold combinations for the three antigens with R (version 3.6.0) and RStudio (version 1.2.1335).	RStudio suggested: (RStudio, RRID:SCR_000432)

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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 this study are related to the sample material used in the validation process of FMIA. In essence, the thresholds and DPOs described here were optimised for the detection of previous infections with mild to moderate symptoms. Despite this, the S-IgG levels correlated strongly with NAb titers. Our results also suggest that FMIA is better at recognising samples with NAbs than many commercial assays [12]. Whilst S-IgG FMIA identified all samples with NAbs, it does not necessarily measure antibodies against neutralising epitopes only, as some samples negative in MNT were considered positive for S-IgG. We conclude that FMIA is valuable in pre-screening of serum samples prior to confirmatory MNT. As the anti-SARS-CoV-2 antibody levels have been found to correlate with the disease severity [23–25], we can expect FMIA to perform well in the serological diagnostics of also previous severe infections. Importantly, while the present data show an excellent sensitivity for IgG-FMIA until 150 DPO, its performance after that remains to be investigated. Estimates of the persistence of immunity to COVID-19 appear to depend on the serological assay used [11,26]. We recently reported that six and twelve months after SARS-CoV-2 infection 98% and 97 % of the patients, respectively, still had S-IgG in FMIA [27]. Other recent studies have found that anti-spike IgG antibodies persist in 90% of individuals seven [28] and nine months [29] after a confirmed SARS-CoV-2 infection. We found ...

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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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   Results from scite Reference Check: We found no unreliable references.

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3. 

   Version published to 10.1101/2021.07.28.21260990v1 on medRxiv

   Jul 30, 2021