Development of robust, indigenous ELISA for detection of IgG antibodies against CoV-2 N and S proteins: mass screening

Srivastava, Ashish Kumar; Gupta, Avinash; Chauhan, Deepika; Meena, R. C.; Sugadev, R.; Eslavath, Malleswara Rao; Gupta, Harshita; Karuna,; Singh, Sayar; Singh, Yamini; Tiwari, Ruchi; Kohli, Veena; Varshney, Rajeev K.; Ganju, Lilly

doi:10.1007/s00253-022-12113-8

Cited by 3 publications

(1 citation statement)

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“…In this study, we optimised and validated an in-house SARS-CoV-2 spike, RBD and nucleoprotein IgG, IgM and IgA binding antibody ELISA that is relevant for serosurveillance studies in populations primarily comprised of asymptomatic and mildly symptomatic COVID-19 cases ( 29 ). Previously, similar efforts ( 30 – 32 ) to develop SARS-CoV-2 binding antibody ELISAs have been limited by the heavy reliance on hospitalised patients with severe disease as the positive controls in determining cut-off threshold values. Such criteria are sub-optimal in Sub-Saharan Africa and similarly affected regions, where SARS-CoV-2 infection has mostly been asymptomatic or accompanied by mild symptoms because the resulting threshold values would be so stringent as to preclude detection of mild infection, leading to a high false-negativity rate ( 33 ).…”

Section: Discussionmentioning

confidence: 99%

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

Oluka¹,

Namubiru²,

Kato³

et al. 2023

Front. Immunol.

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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.

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