Methodological approaches to optimize multiplex oral fluid SARS-CoV-2 IgG assay performance and correlation with serologic and neutralizing antibody responses
Methodological approaches to optimize multiplex oral fluid SARS-CoV-2 IgG assay performance and correlation with serologic and neutralizing antibody responses
ABSTRACT BackgroundOral fluid (hereafter, saliva) is a non-invasive and attractive alternative to blood for SARS-CoV-2 IgG testing; however, the heterogeneity of saliva as a matrix poses challenges for immunoassay performance. ObjectivesTo optimize performance of a magnetic microparticle-based multiplex immunoassay (MIA) for SARS-CoV-2 IgG measurement in saliva, with consideration of: i) threshold setting and validation across different MIA bead batches; ii) sample qualification based on salivary total IgG concentration; iii) calibration to U.S. SARS-CoV-2 serological standard binding antibody units (BAU); and iv) correlations with blood-based SARS-CoV-2 serological and neutralizing antibody (nAb) assays. MethodsThe salivary SARS-CoV-2 IgG MIA included 2 nucleocapsid (N), 3 receptor-binding domain (RBD), and 2 spike protein (S) antigens. Gingival crevicular fluid (GCF) swab saliva samples were collected before December, 2019 (n=555) and after molecular test-confirmed SARS-CoV-2 infection from 113 individuals (providing up to 5 repeated-measures; n=398) and used to optimize and validate MIA performance (total n=953). Combinations of IgG responses to N, RBD and S and total salivary IgG concentration (μg/mL) as a qualifier of nonreactive samples were optimized and validated, calibrated to the U.S. SARS-CoV-2 serological standard, and correlated with blood-based SARS-CoV-2 IgG ELISA and nAb assays. ResultsThe sum of signal to cutoff (S/Co) to all seven MIA SARS-CoV-2 antigens and disqualification of nonreactive saliva samples with ≤15 μg/mL total IgG led to correct classification of 62/62 positives (sensitivity [Se]=100.0%; 95% confidence interval [CI]=94.8%, 100.0%) and 108/109 negatives (specificity [Sp]=99.1%; 95% CI=97.3%, 100.0%) at 8-million beads coupling scale and 80/81 positives (Se=98.8%; 95% CI=93.3%, 100.0%] and 127/127 negatives (Sp=100%; 95% CI=97.1%, 100.0%) at 20-million beads coupling scale. Salivary SARS-CoV-2 IgG crossed the MIA cutoff of 0.1 BAU/mL on average 9 days post-COVID-19 symptom onset and peaked around day 30. Among n=30 matched saliva and plasma samples, salivary SARS-CoV-2 MIA IgG levels correlated with corresponding-antigen plasma ELISA IgG (N: ρ=0.67, RBD: ρ=0.76, S: ρ=0.82; all p<0.0001). Correlations of plasma SARS-CoV-2 nAb assay area under the curve (AUC) with salivary MIA IgG (N: ρ=0.68, RBD: ρ=0.78, S: ρ=0.79; all p<0.0001) and with plasma ELISA IgG (N: ρ=0.76, RBD: ρ=0.79, S: ρ=0.76; p<0.0001) were similar. ConclusionsA salivary SARS-CoV-2 IgG MIA produced consistently high Se (>98.8%) and Sp (>99.1%) across two bead coupling scales and correlations with nAb responses that were similar to blood-based SARS-CoV-2 IgG ELISA data. This non-invasive salivary SARS-CoV-2 IgG MIA could increase engagement of vulnerable populations and improve broad understanding of humoral immunity (kinetics and gaps) within the evolving context of booster vaccination, viral variants and waning immunity.
Kruczynski Kate、Pekosz Andrew、Clarke William、Heaney Christopher D.、Pisanic Nora、Thomas David L.、Antar Annukka A. R.、Detrick Barbara、Rivera Magdielis Gregory、Dhakal Santosh、Betenbaugh Michael J.、Spicer Kristoffer、Klein Sabra L.、Randad Pranay R.、Manabe Yukari C.
Department of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins UniversityDepartment of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins University||Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins UniversityDepartment of Pathology, School of Medicine, Johns Hopkins UniversityDepartment of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins University||Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins University||Department of International Health, Bloomberg School of Public Health, Johns Hopkins UniversityDepartment of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins UniversityDivision of Infectious Diseases, Department of Medicine, School of Medicine, Johns Hopkins University||Department of Pathology, School of Medicine, Johns Hopkins UniversityDivision of Infectious Diseases, Department of Medicine, School of Medicine, Johns Hopkins UniversityDepartment of Pathology, School of Medicine, Johns Hopkins University||Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins UniversityDepartment of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins UniversityDepartment of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins UniversityDepartment of Chemical and Biomolecular Engineering, Whiting School of Engineering, Johns Hopkins UniversityDepartment of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins UniversityDepartment of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University||Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins UniversityDepartment of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins UniversityDivision of Infectious Diseases, Department of Medicine, School of Medicine, Johns Hopkins University||Department of Pathology, School of Medicine, Johns Hopkins University
医学研究方法基础医学预防医学
SARS-CoV-2COVID-19salivaoral fluidantibody kineticsseroprevalenceserosurveillancemultiplex assay
Kruczynski Kate,Pekosz Andrew,Clarke William,Heaney Christopher D.,Pisanic Nora,Thomas David L.,Antar Annukka A. R.,Detrick Barbara,Rivera Magdielis Gregory,Dhakal Santosh,Betenbaugh Michael J.,Spicer Kristoffer,Klein Sabra L.,Randad Pranay R.,Manabe Yukari C..Methodological approaches to optimize multiplex oral fluid SARS-CoV-2 IgG assay performance and correlation with serologic and neutralizing antibody responses[EB/OL].(2025-03-28)[2025-05-25].https://www.medrxiv.org/content/10.1101/2022.12.22.22283858.点此复制
评论