Evaluation of isotype specific salivary antibody assays for detecting previous SARS-CoV-2 infection in children and adults

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. ### Competing Interest Statement AF is a member of the Joint Committee on Vaccination and Immunisation, the UK national immunisation technical advisory group and is chair of the WHO European regional technical advisory group of experts (ETAGE)on immunisation and ex officio a member of the WHO SAGE working group on COVID vaccines. He is investigator on studies and trials funded by Pfizer, Sanofi, Valneva, the Gates Foundation and the UK government. ### Funding Statement We acknowledge funding support from The University of Bristol and the Elizabeth Blackwell Institute supported by Bristol Alumni and Friends for equipment and reagents to conduct assay development and test accuracy studies. Deployment of assays to household outbreaks in the CoMMinS study was supported by the MRC [MR/V028545/1]. AT is supported by the Wellcome Trust (217509/Z/19/Z) and UKRI through the JUNIPER consortium MR/V038613/1 and CoMMinS study MR/V028545/1. E.B.P. was partly supported by the NIHR Health Protection Research Unit (HPRU) in Behavioural Science and Evaluation. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. The NIHR had no role in writing the manuscript or the decision to publish it. E.B.P. is funded via the JUNIPER Consortium (MRC grant no. MR/V038613/1) and MRC grant no. MC/PC/19067. NJT is a Wellcome Trust Investigator (202802/Z/16/Z), is the PI of the Avon Longitudinal Study of Parents and Children (MRC & WT 217065/Z/19/Z), is supported by the University of Bristol NIHR Biomedical Research Centre (BRC-1215-2001), the MRC Integrative Epidemiology Unit (MC\_UU\_00011/1) and works within the CRUK Integrative Cancer Epidemiology Programme (C18281/A29019). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Whole saliva from healthy donors (pre- and during the COVID-19 pandemic) was obtained via the Bristol BioBank (NHS REC 20/WA/0273) under the use application U-0042. Pre-pandemic (PP) sample cohorts were obtained in two ways. PP cohort 1 samples were collected in Portugal under local Ethics for a specific research study, remaining samples were stored and used for this work under NHS REC 13/NW/0439. PP cohorts 2-5 were collected under further Bristol BioBank deposit applications, and upon study completion these sample sets were deposited into the Bristol BioBank and released to this project under use application U-0042. Saliva samples were collected from household outbreaks during the CoMMinS study under NHS REC 20/HRA/4876. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. Machine learning analysis is available as a Jupyter notebook at https://github.com/Bristol-UNCOVER/Saliva\_data\_ML\_analysis/blob/main/Saliva\_dataset_analysis.ipynb. [https://github.com/Bristol-UNCOVER/Saliva\_data\_ML\_analysis/blob/main/Saliva\_dataset_analysis.ipynb][1] [1]: https://github.com/Bristol-UNCOVER/Saliva_data_ML_analysis/blob/main/Saliva_dataset_analysis.ipynb