TY - JOUR
T1 - Methodological approaches to optimize multiplex oral fluid SARS-CoV-2 IgG assay performance and correlation with serologic and neutralizing antibody responses
AU - Pisanic, Nora
AU - Antar, Annukka A.R.
AU - Kruczynski, Kate L.
AU - Gregory Rivera, Magdielis
AU - Dhakal, Santosh
AU - Spicer, Kristoffer
AU - Randad, Pranay R.
AU - Pekosz, Andrew
AU - Klein, Sabra L.
AU - Betenbaugh, Michael J.
AU - Detrick, Barbara
AU - Clarke, William
AU - Thomas, David L.
AU - Manabe, Yukari C.
AU - Heaney, Christopher D.
N1 - Publisher Copyright:
© 2023
PY - 2023/3
Y1 - 2023/3
N2 - Background: Oral 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. Objectives: To 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. Methods: The 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. Results: The 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 = 0.76, RBD: ρ = 0.83, S: ρ = 0.82; all p < 0.001). 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 < 0.001) and with plasma ELISA IgG (N: ρ = 0.76, RBD: ρ = 0.79, S: ρ = 0.76; p < 0.001) were similar. Conclusions: A 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.
AB - Background: Oral 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. Objectives: To 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. Methods: The 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. Results: The 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 = 0.76, RBD: ρ = 0.83, S: ρ = 0.82; all p < 0.001). 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 < 0.001) and with plasma ELISA IgG (N: ρ = 0.76, RBD: ρ = 0.79, S: ρ = 0.76; p < 0.001) were similar. Conclusions: A 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.
KW - Antibody kinetics
KW - COVID-19
KW - Multiplex assay
KW - Oral fluid
KW - SARS-CoV-2
KW - Saliva
KW - Seroprevalence
KW - Serosurveillance
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U2 - 10.1016/j.jim.2023.113440
DO - 10.1016/j.jim.2023.113440
M3 - Article
C2 - 36773929
AN - SCOPUS:85149154513
SN - 0022-1759
VL - 514
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
M1 - 113440
ER -