Evaluation of isotype specific salivary antibody assays for detecting previous SARS-CoV-2 infection in children and adults
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Abstract
Saliva is easily obtainable non-invasively and potentially suitable for detecting both current and previous SARS-CoV-2 infection. We established 6 standardised enzyme linked immunosorbent assays (ELISA) capable of detecting IgA and IgG antibodies to whole SARS-CoV-2 spike protein, to its receptor binding domain region and to nucleocapsid protein in saliva. In test accuracy (n=320), we found that spike IgG performed best (ROC AUC: 95.0%, 92.8-97.3%), followed by spike IgA (ROC AUC: 89.9%, 86.5-93.2%) for discriminating between pre-pandemic and post COVID-19 saliva samples. Using machine learning, diagnostic performance was improved when a combination of tests was used. As expected, salivary IgA was poorly correlated with serum, indicating an oral mucosal response whereas salivary IgG responses were predictive of those in serum. When deployed to 20 household outbreaks undergoing Delta and Omicron infection, antibody responses were heterogeneous but remained a reliable indicator of recent infection. Intriguingly, unvaccinated children showed evidence of exposure almost exclusively through specific IgA responses in the absence of evidence of viral infection. We have provided robust standardisation, evaluation, and field-testing of salivary antibody assays as tools for monitoring SARS-CoV-2 immune responses. Future work should focus on investigating salivary antibody responses following infection and vaccination to understand patterns of SARS-CoV-2 transmission and inform ongoing vaccination strategies.
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SciScore for 10.1101/2022.04.11.22273690: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources Secondary antibodies were used as follows with the dilution factor indicated: HRP conjugated anti-human IgG (Southern Biotech: 1 in 25,000) and IgA (Sigma: 1 in 6,000-10,000). anti-human IgGsuggested: (Aviva Systems Biology Cat# AVARP 00010, RRID:AB_271342)Recombinant DNA Sentences Resources A codon-optimized, N-terminal His6 tagged full length nucleocapsid protein of SARS-CoV-2 was synthesized and cloned by GenScript into a pET28a bacterial expression plasmid, (called here pET28a-NP-FL). pET28asuggested: RRID:Addgene_139598)The pET28a-NP-FL plasmid was transformed into E. coli strain BL21 (DE3) and … SciScore for 10.1101/2022.04.11.22273690: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources Secondary antibodies were used as follows with the dilution factor indicated: HRP conjugated anti-human IgG (Southern Biotech: 1 in 25,000) and IgA (Sigma: 1 in 6,000-10,000). anti-human IgGsuggested: (Aviva Systems Biology Cat# AVARP 00010, RRID:AB_271342)Recombinant DNA Sentences Resources A codon-optimized, N-terminal His6 tagged full length nucleocapsid protein of SARS-CoV-2 was synthesized and cloned by GenScript into a pET28a bacterial expression plasmid, (called here pET28a-NP-FL). pET28asuggested: RRID:Addgene_139598)The pET28a-NP-FL plasmid was transformed into E. coli strain BL21 (DE3) and expressed. pET28a-NP-FLsuggested: NoneSoftware and Algorithms Sentences Resources SARS-CoV-2 N6 gene primers and probes were designed using Primer3 and a consensus multiple sequence alignment of 658 SARS-CoV-2 N gene sequences downloaded from GenBank240. Primer3suggested: (Primer3, RRID:SCR_003139)The AdaBoost algorithm was imported into the notebook from the Python package scikit-learn42, full details on dataset construction, classifier training and performance is given in supplementary information ‘Methods for machine learning analysis’. Pythonsuggested: (IPython, RRID:SCR_001658)Results from OddPub: Thank you for sharing your code and data.
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:Our study has several limitations. We did not evaluate analytical specificity to other seasonal human coronaviruses (HCoVs) nor other respiratory viruses using our large pre-pandemic collection, where presence of antibodies to other confirmed coronaviruses may account for some false-positive results21. However, anti-spike salivary antibody responses have been demonstrated to be highly specific by others9,31. When we performed the test accuracy aspects of this study, we were unable to obtain 200 samples from recovered PCR-confirmed individuals as per MHRA guidelines, so estimates of test sensitivity are uncertain32. Finally, deployment of the best performing anti-spike assays for salivo-surveillance in vaccinated populations presents challenges: these assays cannot distinguish infected from vaccinated individuals, while anti-N-protein salivo-conversion appears to occur infrequently in infected individuals. Nonetheless, we did observe clear increases in salivo-positivity following infection in vaccinated individuals (in the household study), offering a potential means to identify periods of transmission when deployed in a mixed population. Our findings emphasise the need for further work on understanding factors associated with SARS-CoV-2 mucosal antibody profiles and the heterogeneity in responses observed. Ongoing monitoring of mucosal antibody responses is essential for understanding transmission of SARS-CoV-2 and informing vaccination strategies, especially if future candid...
Results from TrialIdentifier: No clinical trial numbers were referenced.
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Results from scite Reference Check: We found no unreliable references.
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