Optimisation and Validation of a conventional ELISA and cut-offs for detecting and quantifying anti-SARS-CoV-2 Spike, RBD, and Nucleoprotein IgG, IgM, and IgA antibodies in Uganda

Abstract

There is an urgent need for better immunoassays to measure antibody responses as part of immune-surveillance activities and to profile immunological responses to emerging SARS-CoV-2 variants. We optimised and validated an in-house conventional ELISA to identify and quantify SARS-CoV-2 spike- (S-), receptor binding domain- (RBD-), and nucleoprotein- (N-) directed IgG, IgM, and IgA binding antibodies in the Ugandan population and similar settings. Pre- and post-pandemic specimens were used to compare the utility of mean ± 2SD, mean ± 3SD, 4-fold above blanks, bootstrapping, and receiver operating characteristic (ROC) analyses in determining optimal cut-off optical densities at 450 nm (OD) for discriminating between antibody positives and negatives. “Limits of detection” (LOD) and “limits of quantitation” (LOQ) were validated alongside the assay’s uniformity, accuracy, inter-assay and inter-operator precision, and parallelism. With spike-directed sensitivity and specificity of 95.33 and 94.15%, respectively, and nucleoprotein sensitivity and specificity of 82.69 and 79.71%, ROC was chosen as the best method for determining cutoffs. Accuracy measurements were within the expected CV range of 25%. Serum and plasma OD values were highly correlated (r = 0.93, p=0.0001). ROC-derived cut-offs for S-, RBD-, and N-directed IgG, IgM, and IgA were 0.432, 0.356, 0.201 (S), 0.214, 0.350, 0.303 (RBD), and 0.395, 0.229, 0.188 (N). The sensitivity and specificity of the S-IgG cut-off were equivalent to the WHO 20/B770-02 S-IgG reference standard at 100% level. Spike negative IgG, IgM, and IgA ODs corresponded to median antibody concentrations of 1.49, 3.16, and 0 BAU/mL, respectively, consistent with WHO low titre estimates. Anti-spike IgG, IgM, and IgA cut-offs were equivalent to 18.94, 20.06, and 55.08 BAU/mL. For the first time, we provide validated parameters and cut-off criteria for the in-house detection of subclinical SARS-CoV-2 infection and vaccine-elicited binding antibodies in the context of Sub-Saharan Africa and populations with comparable risk factors.

Rapid, early, and potent Spike-directed IgG, IgM, and IgA distinguish asymptomatic from mildly symptomatic COVID-19 in Uganda, with IgG persisting for 28 months

“…Spike-, RBD-, and N-directed IgG, IgM, and IgA antibodies were quantified using an in-house ELISA adapted from Pickering et al. ( 37 ), optimized and validated for use in this largely asymptomatic or mildly symptomatic COVID-19infected population ( 38 ). Briefly, 96-well flat-bottomed medium-binding plates (Greiner Bio-One, #655001) coated with 50 μl of N-, RBD-, or S-Protein antigens based on the wildtype prototype strain (R&D Systems #10474-CV-01M, #10549-CV-01M) at three µg/ml (0.15µg per well) in PBS were incubated overnight at 4°C.…”

“…Blank well OD values were subtracted from those in specimen wells to obtain the net response. Receiver operator characteristic (ROC) analysis derived cut-offs for S-, RBD-, and N-directed IgG, IgM, and IgA optical densities were 0.432, 0.356, 0.201(S protein), 0.214, 0.350, 0.303 (RBD), and 0.395, 0.229, 0.188 (N protein), respectively, as described elsewhere ( 38 ).…”

Pre-pandemic SARS-CoV-2-specific IFN-γ and antibody responses were low in Ugandan samples and significantly reduced in HIV-positive specimens

“…Here, we used a validated in-house ELISA that uses the complete nucleoprotein proteome, with all test specimens first prediluted 100-fold to define a virus-specific response. The assay limits of detection and cut-off thresholds used were established and validated for the Ugandan population ( 19 ). The simultaneous ELISpot screening of our specimens using seven SARS CoV-2 antigenic regions to establish a comparable human coronavirus T-cell cross-reactivity prevalence of 27.9% corroborates our antibody prevalence data.…”

“…SARS-CoV-2-specific S and N binding IgG optical densities (450nm) and concentrations (ng/ml) were quantified using an in-house optimized conventional ELISA, as described before ( 19 ). Briefly, 96-well flat-bottomed membrane-binding plates (Greiner Bio-One) coated with 3 μg/ml recombinant S or N protein in PBS were incubated with serum specimens diluted 1:100 in PBS-T containing 1% Bovine Serum Albumin (BSA).…”

Source Tracing of Kidney Injury via the Multispectral Fingerprint Identified by Machine Learning-Driven Surface-Enhanced Raman Spectroscopic Analysis

“…The limit of detection (LOD) of 120.1 fM was calculated by the limit of detection formula (LOD = 3.3 × SD/K). 45 To eliminate residual chemicals, FBS NCAs were treated with 0.1 M KI, effectively displacing impurities and reducing interference in SERS measurements, resulting in a clean substrate (Figure 1L). Characterization of Exosomes.…”