key: cord-0330984-m83h5y2g authors: Hoschler, K.; Ijaz, S.; Andrews, N.; Ho, S.; Dicks, S.; Jegatheesan, K.; Poh, J.; Warrener, L.; Kankeyan, T.; Baawuah, F.; Beckmann, J.; Okike, I. O.; Ahmad, S.; Garstang, J.; Brent, A. J.; Brent, B.; Aiano, F.; Brown, K. E.; Ramsay, M. E.; Brown, D. W.; Parry, J. V.; Ladhani, S. N.; Zambon, M. title: SARS-CoV-2 IgG detection in human oral fluids date: 2021-07-07 journal: nan DOI: 10.1101/2021.07.07.21260121 sha: f170bdd40d7ab7429744e2136a36ae94646c8730 doc_id: 330984 cord_uid: m83h5y2g Seroepidemiological studies to monitor antibody kinetics are important for assessing the extent and spread of SARS-CoV-2 in a population. Non-invasive sampling methods are advantageous to reduce the need for venepuncture, which may be a barrier to investigations particularly in paediatric populations. Oral Fluids are obtained by gingiva-crevicular sampling from children and adults and are very well accepted. ELISA based on these samples have acceptable sensitivity and specificity compared to conventional serum-based antibody ELISAs and are suitable for population-based surveillance. We describe the development and evaluation of SARS-COV-2 IgG ELISAs using SARS-CoV-2 viral nucleoprotein (NP) and spike (S) proteins in IgG isotype capture format and an indirect receptor-binding-domain (RBD) IgG ELISA, intended for use in children. All three assays were assessed using a panel of 1999 paired serum and oral fluids from children and adults participating in national primary school SARS-CoV-2 surveillance studies during and after the first and second pandemic wave in the UK. The anti NP IgG capture assay was the best candidate, with an overall sensitivity of 75% (95% CI: 71-79%) specificity of 99% (95% CI: 78-99%) when compared with paired serum antibodies measured using a commercial assay SARS-CoV-2 nucleoprotein IgG assay (Abbott, Chicago, IL, USA). Higher sensitivity was observed in children (80%, 95% CI: 71-88%) compared to adults (67%, CI: 60%-74%). Oral fluid assays using spike protein and RBD antigens were also 99% specific and achieved reasonable but lower sensitivity in the target population (78%, 95% CI (68%-86%) and 53%, 95% CI (43%-64%), respectively). PHE initiated SARS-CoV-2 surveillance in primary schools across England (6). In total, 131 schools 101 across England were recruited; 86 schools provided weekly nasal swabs for SARS-CoV-2 RT-PCR 102 and 45 schools provided a blood sample, nasal swab and an oral fluid sample at the beginning and 103 end of the autumn term in 2020 (6), providing population based materials to assess the feasibility and 104 performance of oral fluid tests. In this study, we evaluated three different in-house enzyme 5 Headteachers in participating primary schools sent the study information pack to parents and staff at 119 the start of the study and those interested in taking part were asked to sign a consent form and 120 complete a short questionnaire. PHE investigators attended the school premises in the period from 121 28th May -10th July 2020 and took nasal swabs and blood samples from participating children and 122 provided guidance and supervision of the oral fluid self-sampling (Table S1 , Supplementary 123 information). The oral fluids were collected on the same day as the paired venous blood, using the Oracol™ foam 125 swab. This collects gingiva-crevicular fluid when brushing the gum line for 2 minutes, after which the 126 swab is re-inserted into a plastic container for transportation. Samples are stable for transport at 127 ambient temperature (14, 15). All samples were couriered to PHE Colindale for same-day processing 128 and storage. On receipt in the laboratory, OF was extracted from the foam swab using 1ml of an elution buffer 131 (Phosphate buffered saline containing 10% Foetal Calf serum and 250ug/ml Gentamicin and 0.5ug/ml 132 Fungizone), The swab tube was centrifuged at 3000 g for 5 minutes in bench top centrifuge to remove 133 cellular debris and, the swab removed and discarded. Samples were stored at -20C prior to testing. The cut-off value for OF samples for all assays was determined by age group (children and adults) 172 from the serum-OF pairs, using exploratory sensitivity versus specificity analysis (see supplement 173 Figure S1 and Table S2 ) with the result from the commercial serum test considered the true result. The final cut-off was the value with the highest possible sensitivity retaining the specificity minimum of All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; 7 Statistical analysis confidence intervals. This was done at a range of cut-offs by ROC-curve analysis which informed on the optimal cut-offs. The area under the ROC curve was also calculated with 95% confidence 186 intervals as an overall measure of assay performance. Assays were compared visually using scatter 187 plots with logged scaled axes and lines of best fit added and the index of multiple correlation 188 calculated (R2). This was also done to compare results to total IgG. Total IgG was compared between 189 those positive and negative by Abbott on serum using Tobit regression. Paired serum and OF samples from 1,999 subjects were tested (Table S1) shows between 58% and 65% similarity to NP of seasonal coronaviruses, and cross-reactivity has 248 been observed in multiple studies (19, 20). The S protein, specifically the RBD region of this protein, 249 is the target for neutralising antibodies and, antibodies against the S1 subunit of the spike protein or For OF sampling, the IgG content of the extracted specimen represents on average a dilution of 253 approximately 1/1,000 of that in blood plasma. As a result, antibody tests using OFs tend to have 254 lower sensitivity than those designed for serum, but this analyte is successfully used in 255 seroepidemiology studies for other viruses e.g. measles and HIV (22-24). The concentration of OFs 256 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.07.21260121 doi: medRxiv preprint 9 varies between individual specimens, ranging from <0.5mg/L to >30mg/L (14). This variability in IgG concentrations makes the choice of assay format more critical. Generally, GICAP antibody tests have proved to be most robust when detecting viral antibodies in OFs as they are tolerant to the very wide 259 range of IgG concentration. The strength of any reactivity in a capture assay is dependent on the 260 proportion of total IgG that is specific for the target antigen, in this case, the SARS-CoV-2 viral the solid phase, the quantity of captured antibodies, while only a part of the total IgG captured, will be 263 constant. In our study, measurement of total IgG content to assess OF sample quality revealed a 264 strong correlation with RBD ELISA reactivity, but not with NP or S protein ELISAs. This reflects the As anticipated, overall sensitivity of detection of SARS CoV-2 IgG OF antibodies was lower than 292 serum. However, we judge that all three in-house assays were sufficiently accurate for large-scale 293 use in population-based studies, with statistical adjustment. Taking into consideration the three OF 294 assays and, the reproducibility, robustness, reagent supply chain and sustainability of service delivery, the IgG isotype specific SARS-CoV-2 NP capture EIA has been adopted as the principal test 296 for the OF-based SARS-CoV-2 antibody surveillance studies in children. A limitation of using a single 297 antigen assay, however, is that antibody kinetics may vary over time. Following mild SARS-CoV-2 298 infection, for example, serum nucleoprotein antibodies decline more rapidly than spike protein 299 antibodies (19), but this may differ for severe illness and may be also dependent on the assays used 300 (30). Additionally, little is known about antibody kinetics in children, which may differ over time since 301 infection. It is, therefore, possible that exclusive use of the NP capture assay may need to be re- All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. Dotted lines represent data trends in each assay. The detection limit for the total IgG determination is 15mg/mldata points from samples with IgG All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.07.21260121 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.07.21260121 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.07.21260121 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.07.21260121 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. COVID-19 314 pulmonary pathology: a multi-institutional autopsy cohort from Italy and New York City Multiorgan 317 impairment in low-risk individuals with post-COVID-19 syndrome: a prospective, community-based 318 study SARS-CoV-2: the Mount Sinai COVID-19 autopsy experience Covid-19 in 322 children: A brief overview after three months experience SARS-CoV-2 infection and 11 Oral fluid testing during 14 A comparison of oral fluid collection devices for use in the 2 66