Mapping of SARS-CoV-2 IgM and IgG in gingival crevicular fluid: Antibody dynamics and linkage to severity of COVID-19 in hospital inpatients

Abstract

•Gingival crevicular fluid forms a useful analyte for detecting SARS-CoV-2 antibody.•There are differential patterns of antibody reactivity across NP and Spike.•Higher antibody levels in early acute phase linked to severity of COVID-19. IntroductionThe use of non-venous analytes has been a priority public health tool for the diagnosis, monitoring and surveillance of a range of pathogens. These methods, primarily based on Dried Blood Spots (DBS) and Gingival Crevicular Fluid (GCF), were first developed in the UK and have been applied to investigate outbreak and transmission events,1Haywood B. Tedder R.S. Beebeejaun K. Balogun K. Mandal S. Andrews N. et al.Oral fluid testing facilitates understanding of hepatitis A virus household transmission.Epidemiol Infect. 2019; 147: e105Crossref PubMed Scopus (1) Google Scholar,2Glynn J.R. Bower H. Johnson S. Houlihan C.F. Montesano C. Scott J.T. et al.Asymptomatic infection and unrecognised Ebola virus disease in Ebola-affected households in Sierra Leone: a cross-sectional study using a new non-invasive assay for antibodies to Ebola virus.Lancet Infect Dis. 2017; 17: 645-653Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar to improve the diagnosis of infections in underserved populations3Craine N. Parry J. O'Toole J. D'Arcy S. Lyons M. Improving blood-borne viral diagnosis; clinical audit of the uptake of dried blood spot testing offered by a substance misuse service.J Viral Hepat. 2009; 16: 219-222Crossref PubMed Scopus (34) Google Scholar,4Dodds J.P. Johnson A.M. Parry J.V. Mercey D.E. A tale of three cities: persisting high HIV prevalence, risk behaviour and undiagnosed infection in community samples of men who have sex with men.Sex Transm Infect. 2007; 83: 392-396Crossref PubMed Scopus (54) Google Scholar in addition to monitoring infection trends and informing on the impact of interventions.5Bardsley M. Heinsbroek E. Harris R. Croxford S. Edmundson C. Hope V. et al.The impact of direct-acting antivirals on hepatitis C viraemia among people who inject drugs in England; real-world data 2011–2018.J Viral Hepat. 2021; 28: 1052-1463Crossref Scopus (3) Google Scholar,6Judd A. Hickman M. Hope V.D. Sutton A.J. Simson G.V. Ramsay M.E. et al.Twenty years of selective hepatitis B vaccination: is hepatitis B declining among injecting drug users in England and Wales?.J Viral Hepat. 2007; 14: 584-591Crossref PubMed Scopus (19) Google ScholarThe ability to use non-venous analytes for antigen, antibody and nucleic acid detection and characterisation has applications to answer questions linked to SARS-CoV-2 infections. The virus has been shown to transmit efficiently between individuals and within communities,7Miller E. Waight P.A. Andrews N.J. McOwat K. Brown K.E. Höschler K. et al.Transmission of SARS-CoV-2 in the household setting: a prospective cohort in children and adults in England.J Infect. 2021; 83: 483-489Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 8Adam D.C. Wu P. Wong J.Y. Lau E.H.Y. Tsang T.K. Cauchemez S. et al.Clustering and superspreading potential of SARS-CoV-2 infections in Hong Kong.Nat Med. 2020; 26: 1714-1719Crossref PubMed Scopus (280) Google Scholar, 9Payne D.C. 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Vusirikala A. et al.Infection and transmission of SARS-CoV-2 in London care homes reporting no cases or outbreaks of COVID-19: prospective observational cohort study, England 2020.Lancet Reg Health Eur. 2021; 3100038PubMed Google Scholar, 11Ye F. Xu S. Rong Z. Xu R. Liu X. Deng P. Liu H. Xu X. Delivery of infection from asymptomatic carriers of COVID-19 in a familial cluster.Int J Infect Dis. 2020; 94: 133-138Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 12Zhou R. Li F. Chen F. Liu H. Zheng J. Lei C. Wu X. Viral dynamics in asymptomatic patients with COVID-19.Int J Infect Dis. 2020; 96: 288-290Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 13Putallaz P. Senn L. Bosshard W. Büla C.J. COVID-19 outbreak at a geriatric rehabilitation facility: the silent threat of asymptomatic patients with high viral loads.Geriatrics. 2021; 6 (Basel): 95Crossref PubMed Scopus (1) Google Scholar, 14Glenet M. Lebreil A.L. Heng L. N'Guyen Y. Meyer I. Andreoletti L. Asymptomatic COVID-19 adult outpatients identified as significant viable SARS-CoV-2 shedders.Sci Rep. 2021; 11: 20615Crossref PubMed Scopus (4) Google Scholar The ease of sampling with non-venous analytes and the ability for self-collection allows for rapid and accessible individual and population-based diagnostics and monitoring of prevalence. The convenience and acceptability of sample collection permit population prevalence studies, for example in school children or in immunised self-isolating individual populations, providing an important mechanism for generating data for virus surveillance with a potential to inform policy. A key role of population antibody testing would be to characterise the relationship between the development and dynamics of antibody responses to infection, vaccination, and the impact of the measures on subsequent rates of transmission.Enzyme linked immunoassays formatted for immunoglobulin (Ig) capture onto the solid phase are favoured for the analysis of non-venous analytes. In this study, we compare the application of Immunoglobulin class M (IgM) and Immunoglobulin class G (IgG) Ig-capture assays to detect antibody against the Nucleoprotein and components of the Spike protein on paired GCF and sera to characterise the acute and early convalescent antibody responses in hospitalised patients with COVID-19 in the UK, and correlate antibody reactivity with severity of disease.Materials and methodsStudy setting and approvalsThe International Severe Acute Respiratory and Emerging Infections Consortium (ISARIC) WHO Clinical Characterisation Protocol UK (CCP-UK) study is an ongoing, prospective cohort study recruiting inpatients with COVID-19 from 348 hospital sites in England, Wales, Scotland, and Northern Ireland.15Docherty A.B. Harrison E.M. Green C.A. Hardwick H.E. Pius R. Norman L. et al.Semple MG, ISARIC4C Investigators. Features of 20 133 UK patients in hospital with COVID-19 using the ISARIC WHO clinical characteristics protocol: prospective observational cohort study.BMJ. 2020; 369: m1985Crossref PubMed Scopus (1574) Google Scholar Ethics approval was granted by the South Central—Oxford C Research Ethics Committee in England (13/SC/0149), the Scotland A Research Ethics Committee (20/SS/0028), and the WHO Ethics Review Committee (RPC571 and RPC572; April 2013). The study protocol and further details are available at https://isaric4c.net/protocols/.15Docherty A.B. Harrison E.M. Green C.A. Hardwick H.E. Pius R. Norman L. et al.Semple MG, ISARIC4C Investigators. Features of 20 133 UK patients in hospital with COVID-19 using the ISARIC WHO clinical characteristics protocol: prospective observational cohort study.BMJ. 2020; 369: m1985Crossref PubMed Scopus (1574) Google ScholarStudy participantsRecruitment procedures for the ISARIC WHO CCP-UK study have been described previously.15Docherty A.B. Harrison E.M. Green C.A. Hardwick H.E. Pius R. Norman L. et al.Semple MG, ISARIC4C Investigators. Features of 20 133 UK patients in hospital with COVID-19 using the ISARIC WHO clinical characteristics protocol: prospective observational cohort study.BMJ. 2020; 369: m1985Crossref PubMed Scopus (1574) Google Scholar Briefly, baseline demographic information and key clinical data including patient characteristics, symptom onset dates, illness severity, level of respiratory support, COVID-19-specific treatment and outcome were recorded in case report forms on a REDCap database. Samples included in this study were collected between March 2020 and June 2020 and were from 115 inpatients across five hospitals. A breakdown of patient numbers from hospitals 1 to 5 were as follows n = 53, n = 15, n = 8, n = 24 and n = 15, respectively. With consent, biological samples, including GCF and sera, were collected from recruited patients on days 1, 3, 9 and 28 post-enrolment. Samples from all four time-points were not available for all patients due to prioritisation of delivery of care over research activity early in infection or later hospital discharge or death. Only patients with matched GCF and serum samples were included in this analysis. Patients with missing data on symptom onset date were excluded. Collated data were analysed with overall trends presented.Patients were grouped into five categories of peak illness severity based on the World Health Organization (WHO) COVID-19 ordinal scale16WHO R&D Blueprint. Novel Coronavirus, COVID-19 Therapeutuc Trial Synopsis. February 18, 2020. Available at:https://cdn.who.int/media/docs/default-source/blue-print/covid-19-therapeutic-trial-synopsis.pdf?sfvrsn=44b83344_1&download=true. Accessed June 2022Google Scholar: (i) no oxygen requirement (WHO score 3); (ii) patient requiring oxygen by face mask or nasal prongs (WHO score 4); (iii) patient requiring high-flow nasal oxygen (HFNO) or non-invasive ventilation (NIV) (WHO score 5); (iv) patients requiring mechanical ventilation (WHO score 6/7) and (v) patients who died in hospital within 28 days of admission (WHO score 8).Collection and extraction of gingival crevicular fluid (GCF) from oracoltm swabsGingival crevicular fluid samples were collected by study staff by brushing Oracol™ S14 foam swabs (Malvern Medical Developments, Worcester, UK) along the upper and lower gumlines, ie the junction between the teeth and the gum, of both sides of a patient's mouth for a total of two minutes. The collected swabs were then frozen at −20 °C. On receipt in the laboratory, the GCF was extracted from the foam swab by adding 1 ml of elution buffer (Phosphate buffered saline containing 10% Foetal Calf Serum, 0.1% Tween-20, 0.5 µg/ml Fungizone and 250 µg/ml Gentamicin). The elution buffer was then moved through the foam by squeezing and agitating the swab against the swab tube wall for approximately 30 s. The swab was then placed directly into the open cap and the cap replaced in the swab tube. The swab was centrifuged at 2000 rpm for five minutes, then removed and discarded. The eluted GCF was transferred into a 2 ml Sarstedt™ tube and stored at −20 °C prior to testing.S1, Spike and NP IgM and IgG capture ELISAsImmunoglobulin capture assays for the detection of IgM and IgG were established for three targets: S1, whole Spike and NP. Horseradish peroxidase (HRP) conjugated SARS-CoV-2 full length Spike Glycoprotein (amino acids 1–1211; His-tag) and Nucleoprotein (amino acids 1–149; His-tag) were purchased from The Native Antigen Company (Kidlington, Oxford, UK). The SARS-CoV-2 S1 antigen (amino acid 1–530, C-terminal twin Strep tags)17Rosa A. Pye V.E. Graham C. Muir L. Seow J. Ng K.W. et al.SARS-CoV-2 can recruit a heme metabolite to evade antibody immunity.Sci Adv. 2021; 7: eabg7607Crossref PubMed Scopus (39) Google Scholar was produced and gifted by The Francis Crick Institute and conjugated to HRP using the Bio-Rad LYNX HRP conjugation kit, in accordance with the manufacturer's instructions. All three recombinant proteins were based on the original Victoria lineage.Solid-phase wells (NUNC® Immunomodule, U8 Maxisorp™ wells) were coated with 100μl volumes of (a) Affinipure rabbit anti-human ɣ (Jackson ImmunoResearch, Ely, Cambridgeshire UK) at 5 μg/ml or (b) Affinpure goat anti-human IgM, Fc5µ fragment (Jackson ImmunoResearch, Ely, Cambridgeshire UK) at 2.5 μg/ml in MicroImmune Coating Buffer for ELISA with preservative; (ClinTech, Guildford, UK). Coating was overnight at 2–8 °C, followed by 3 h at 35–37 °C. Wells were then washed with PBS Tween 20 and quenched with MicroImmune Blocking Solution (ClinTech, Guildford, UK) for 3–4 h at 37 °C. Wells were aspirated and stored dry at 4 °C in sealed pouches with desiccant until use.For these Ig capture ELISAs, 100 µl of GCF or 100 µl of a 1:100 dilution of the serum sample in elution buffer were added to the well, incubated for 60 ± 2 min at 37 °C prior to washing and the addition of the conjugate. One hundred microlitres of the HRP-conjugated recombinant protein for each individual assay were added to the microwells. After a further incubation for 60 ± 2 min at 37 °C, the solid phase was washed and 100 µl of TMB substrate added, incubated for 30 ± 2 min at 37 °C, the reaction was then stopped and measured at 450/630 nm. The cut off was calculated as the mean of 4x negative controls +0.1. Samples with a binding ratio (Sample/Cut Off) of ≥1.0 were considered to be antibody reactive. The GCF IgG assays have a specificity of 98% with a sensitivity of 79%, 75% and 70% for S1, NP and whole Spike tests, respectively.18Hoschler K. Ijaz S. Andrews N. Ho S. Dicks S. Jegatheesan K. et al.SARS antibody testing in children: development of oral fluid assays for IgG measurements.Microbiol Spectr. 2022; 10 (Jan 5)e0078621Crossref PubMed Scopus (6) Google Scholar Assay validation data including assignment of test cut off, correlation between GCF and serum and the impact of total IgG in GCF samples have been previously described.18Hoschler K. Ijaz S. Andrews N. Ho S. Dicks S. Jegatheesan K. et al.SARS antibody testing in children: development of oral fluid assays for IgG measurements.Microbiol Spectr. 2022; 10 (Jan 5)e0078621Crossref PubMed Scopus (6) Google ScholarEndemic seasonal coronavirus NP blockingTo mitigate potential issues with cross reactivity in the NP capture assay, four recombinant seasonal coronavirus nucleoproteins (229E, NL63, OC43 and HKU1), produced and gifted by The Francis Crick Institute, were added to the final SARS-CoV-2 NP conjugate. The seasonal NP antigens were added ‘cold’, ie unconjugated, and acted to block non-specific reactivity, a concept that has been previously described for flavivirus serology.19Tedder R.S. Dicks S. Ijaz S. Santiago de Souza N.C. Vincente de Paula A. Levy F. et al.Ushiro lumb I. Modulated Zika virus NS1 conjugate offers advantages for accurate detection of Zika virus specific antibody in double antigen binding and Ig capture enzyme immunoassays.PLoS ONE. 2019; 14e0215708Crossref PubMed Scopus (4) Google Scholar To demonstrate utility, each seasonal coronavirus NP recombinant protein was added individually and also as a four-mix combination at a final concentration of 2.5 µg/ml to the SARS-CoV-2 NP conjugate and tested on pre-pandemic samples reactive in the unblocked SARS-CoV-2 NP assay.ResultsMatched GCF and serum samples were available from 115 inpatients admitted with COVID-19. Patient characteristics are described in Supplementary Table 1. Of these patients, 34% were in severity group 1, 27% in group 2, 9% in group 3, 15% in group 4, and 14% in group 5. As expected, differences between the severity groups were noted with the overall trend for females to have lower disease severity scores than males. A total of 320 matched samples (160 GCF and 160 serum samples) were included in this analysis.Endemic seasonal coronavirus NP blockingSerum samples collected from patients with confirmed SARS-CoV-2 infection remained IgG antibody reactive in the presence of the blocking antigens when endemic seasonal coronavirus nucleoproteins were added either individually or in combination (Table 1). Some reduction in reactivity was noted when compared to the unblocked wells (Table 1). False positive reactivity observed in samples 5 to 9 was efficiently blocked with the addition of the nucleoproteins from coronavirus 229E and NL63 as well as in the test where all four endemic coronavirus nucleoproteins were added (Table 1).Table 1. ‘Blocking’ data on the NP IgG capture assay; OD values shown in bold indicate reactive samples. Each seasonal coronavirus NP was added individually and also as a four mix combination at a final concentration of 2.5 µg/ml to the SARS-CoV-2 NP conjugate and tested on samples identified to be SARS-CoV-2 antibody positive samples (samples 1 to 4) and pre-pandemic samples reactive in the unblocked SARS-CoV-2 NP assay (samples 5 to 14).SamplesNP IgG Capture Assay (OD)No Block229E BlockNL63 BlockOC43 BlockHKU1 BlockAll 4 BlockSARS-CoV-2 SamplesSample 11.1840.9520.9541.0991.0350.926Sample 22.7222.1582.2112.2512.1662.075Sample 31.2121.0771.1421.1721.0641.019Sample 41.2881.2431.2821.2420.9640.981Samples collected pre-pandemicSample 50.2550.0750.0720.2380.2710.061Sample 60.2780.0890.0970.1570.1980.053Sample 70.2880.0740.060.1160.330.051Sample 80.2340.0590.0580.1940.1960.068Sample 90.330.0560.0510.330.3310.055Sample 100.0880.0360.0360.0330.0730.086Sample 110.0530.0370.040.0410.0570.079Sample 120.1370.1010.1020.1140.150.061Sample 130.0910.0580.0630.0670.0920.051Sample 140.0840.0440.0410.0470.1510.087 Open table in a new tab Development of early antibody responses as measured in gingival crevicular fluid collected from hospital inpatientsAccess to samples collected in the 21 days after symptom onset allowed for the mapping of the early antibody responses in GCF samples. The overall trend across the three proteins from day zero through to day 21, was a rise in antibody reactivity levels (Fig. 1; Table 2) and an increase in the number of reactive samples (Table 2). Differential reactivity was noticeable across the proteins with a higher proportion of samples collected in the 14 days after symptom onset being reactive for anti-NP compared to that displayed for antibody to the Spike and S1 antigens. This was noted for both the IgM and IgG class antibodies (Table 2). The data indicated antibodies (IgM with or without IgG) are detectable in GCF within the first seven days from symptom onset (Table 2). Across the two Spike assays, 21–26%, 49–53%, and 78–82% of the samples were IgM reactive and 15–18%, 35–43%, and 56–78% were IgG reactive by day 7, 14 and 21, respectively. For the NP assay, 42%, 65%, and 65% of the samples were IgM reactive and 42%, 67%, and 87% were IgG reactive by day 7, 14 and 21, respectively.Table 2Development of antibody responses as measured against three recombinant proteins, whole spike, NP and S1, in matched GCF and serum samples collected from hospitalised patients. The percentage of samples antibody reactive (IgM and IgG) and median binding ratios (sample OD/cut off) for each analyte is shown across the early stages of infection (days 0–21 post symptom onset) and at >22 days (range: 22–80 days).Days post symptom onsetOral FluidSerumIgMIgGIgMIgGSpikeNPS1SpikeNPS1SpikeNPS1SpikeNPS1% antibody reactive samples0 to 721.142.126.318.442.115.834.250.044.723.742.128.98 to 1449.365.753.035.867.243.964.265.766.750.762.751.515 to 2178.365.282.678.387.056.5100.095.7100.095.795.7100.0> 2256.846.865.671.887.571.884.375.090.675.090.681.2median binding ratios0 to 71.372.111.871.883.811.753.034.475.472.484.952.778 to 142.63.053.822.645.182.926.775.819.564.988.777.2715 to 214.694.508.605.519.004.8110.508.3218.3210.8716.2113.80> 221.750.753.746.4210.505.287.404.8113.9010.2813.7113.10 Open table in a new tab Antibody dynamics in GCF samplesThe trend was for IgM reactivity to peak by week three after symptom onset in all assays. Overall, a more robust IgM response was noted on tests based on S1 and Spike compared to the NP assay. In the 32 samples collected after week three of symptom onset (range: 22–80 days), a decline in IgM reactivity levels was noted for antibody to all three antigens, this decrease being more marked for the NP protein (Fig. 1, Table 2). The overall trend was for IgG antibody reactivity to continue to rise even in the samples collected after day 22 of symptom onset. However, although reactivity in the S1 and Spike based assays did not increase after week three of symptom onset; a rise in median binding ratios was observed with the anti-NP assay.Comparative analysis of binding ratios across the three recombinant proteins indicated a good correlation in the two Spike based assays with both IgM and IgG (Fig. 2A). This relationship was less defined when comparing the NP to the two Spike assays (Fig. 2B,C), in particular for IgM where there was a notable trend for lower reactivity in the NP IgM assay.Fig. 2. X/Y plots demonstrating the correlation of IgM and IgG responses in GCF samples across three proteins; between S1 and Spike (A), NP and Spike (B) and NP and S1 (C). Binding ratios (BR; sample OD/cut off) have been logged.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Antibody levels in GCF stratified by disease severity scoreAntibody binding ratios were mapped against days from symptoms onset (Fig. 3A,C). A broad distribution of antibody binding ratios was noted across the three SARS-CoV-2 protein targets in both the IgM and IgG assays. The data plot was divided into nine grids split by antibody binding ratio levels (Low [L], Medium [M], High [H]) and across the days post symptom onset (Early: 0–14 days, Middle: 15–30 days and Late: 31–50 days). These data were mapped onto the corresponding severity score for each patient to investigate possible associations. Only the initial sample collected from each patient was included in this analysis with the majority of samples falling into the grids representing the early antibody response. The data suggested increased severity of disease severity scores to be linked to those patients whose samples displayed high antibody levels (H), in particular in the first 14 days post symptom onset (Fig. 3B,C). This trend was seen across all three protein targets and also for both IgM and IgG with some suggestion that the association with increased severity to be more marked in the IgM response. This observation was maintained in the middle phase (15–30 days post symptom onset), although not reaching statistical significance in all instances.Fig. 3Distribution of antibody binding ratios across the three protein targets in both the IgM and IgG assays. In panels A (IgM) and C (IgG), the data is divided into nine grids split by binding ratio levels (Low [L], Medium [M], High [H]) and across the observation period (days) post symptom onset (Early, Middle and Late). In panels B (IgM) and D (IgG), violin plots demonstrate relationships between antibody reactivity levels and disease severity demonstrating that earlier in disease higher antibody levels are associated with more severe disease. The first measurement for each patient was included in the analysis and the Kruskal-Wallis statistical test accounting for multiple testing was implemented.View Large Image Figure ViewerDownload Hi-res image Download (PPT)More limited sample numbers in the late phase restricted analysis and interpretation of the data.Antibody dynamics in matched serum samplesTesting of serum samples showed broadly comparable antibody reactivity trends to the matched GCF samples. This was observed for both IgM and IgG assays and across the three proteins (Table 2; Fig. S1). Positive antibody reactivity was shown to occur earlier in serum samples. As expected, antibody reactivity levels were higher in serum than the matched GCF samples (Table 2; Fig. S1). As with GCF samples, differential reactivity was observed between the NP and Spike and S1 assays in serum samples collected in the first 14 days after symptom onset.DiscussionThere has been a rapid evolution of antibody diagnostics over the course of the COVID19 pandemic encompassing a range of assay formats and investigating multiple targets. Whilst serum/plasma samples remain the gold standard for antibody detection, other non-venous analytes including DBS20Turgeon C.T. Sanders K.A. Granger D. Nett S.L. Hilgart H. Matern D. et al.Detection of SARS-CoV-2 IgG antibodies in dried blood spots.Diagn Microbiol Infect Dis. 2021; 101115425Crossref PubMed Scopus (11) Google Scholar, 21Marchand A. Roulland I. 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Padgett W. et al.A multiplex noninvasive salivary antibody assay for SARS-CoV-2 infection and its application in a population-based survey by mail.Microbiol Spectr. 2021; 9e0069321Crossref PubMed Scopus (6) Google Scholar, 25Chiang S.H. Tu M. Cheng J. Wei F. Li F. Chia D. et al.Development and validation of a quantitative, non invasive, highly sensitive and specific, electrochemical assay for anti-SARS-CoV-2 IgG antibodies in saliva.PLoS ONE. 2021; 16e0251342Crossref Scopus (6) Google Scholar have been successfully utilised for the detection and monitoring of antibody responses. Gingival crevicular fluid has some useful advantages over these analytes. The collection of GCF is less invasive than DBS and may be better suited for sample collection in specific populations such as children. In addition, as GCF, obtained as we describe using oral fluid collection swabs, is derived directly by transudation from blood plasma, immunoglobulin levels may be higher when compared to that in saliva.The assays applied in this study are based on antibody capture formats which have been primarily used for antibody detection methods based on GCF.Being proportionality assays they are less affected by variability in sample quality and the quantity of Ig therein contained, a feature which may be associated with the collection of various non-venous analytes. Due to the close homology shared between the NP of SARS-CoV-2 and the NP of endemic seasonal coronaviruses, NP IgM and IgG capture assay based on the concept of blocking were established. The observed blocking patterns were specific as false reactivity detected in five samples collected pre-pandemic was blocked by the alphacoronavirus NP antigens (229E and NL63) but not the betacoronavirus NP antigens (OC43 and HKU1) (Table 1). Some reduction in reactivity was noted in serum samples collected from confirmed SARS-CoV-2 infected patients in the presence of the blocking antigens. However, this was not unexpected due to blocking of NP epitopes common across the coronavirus family. All recombinant proteins in our investigation were based on the original Victoria lineage. This would have been the common SARS-CoV-2 virus circulating at the time the samples included in our investigation were collected. With emerging new variants, consideration will need to be given to the choice of recombinant proteins in particular those based on the S1 with additional verification needed to ensure sensitivity.Mapping of the early antibody response indicated IgM and IgG reactivity to be detectable in GCF samples as early as week one post onset of symptoms with antibody reactivity levels, and the proportion of reactive samples increasing through weeks two and three. The overall trends were mirrored across both GCF and serum analytes demonstrating the utility of GCF for the detection and monitoring of the early antibody response.Differential patterns of antibody reactivity across the NP and the Spike based proteins were noted. Antibody responses to NP were found to appear earlier, a trend noted in both the serum and GCF analytes and for both the IgM and IgG antibody classes. These observations are in line with what has been previously described with serum as the main analyte.26Brochot E. Demey B. Touzé A. Belouzard S. Dubuisson J. Schmit J.L. Anti-spike, anti-nucleocapsid and neutralising antibodies in SARS-CoV-2 inpatients and asymptomatic individuals.Front Microbiol. 2020; 11584251Crossref PubMed Scopus (77) Google Scholar Brochot and colleagues,26Brochot E. Demey B. Touzé A. Belouzard S. Dubuisson J. Schmit J.L. Anti-spike, anti-nucleocapsid and neutralising antibodies in SARS-CoV-2 inpatients and asymptomatic individuals.Front Microbiol. 2020; 11584251Crossref PubMed Scopus (77) Google Scholar examining time to seroconversion, also demonstrated differences in antibody dynamics between various spike proteins. Antibodies targeting the receptor binding domain (anti-RBD) were the earliest to be detected with anti-RBD profiles mirroring the antibody responses to NP. Anti-S2 antibodies followed with a mean time lag of two days; antibodies to the S1 subunit were the last to be detected. The report also no