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

Oluka, G. K., Namubiru, P., Kato, L., Ankunda, V., Gombe, B., Cotten, M. , Musenero, M., Kaleebu, P., Fox, J. and Serwanga, J. (2023) 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. Frontiers in Immunology, 14, 1113194. (doi: 10.3389/fimmu.2023.1113194) (PMID:36999017) (PMCID:PMC10045470)

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.

• Item Type: Articles
• Additional Information: The work was funded by the government of Uganda under the Science, Technology, and Innovation Secretariat-Office of the President (STI-OP), grant number: MOSTI-PRESIDE-COVID-19-2020/15. The work was conducted at the MRC/UVRI and LSHTM Uganda Research Unit which is jointly funded by the UK Medical Research Council (MRC) part of UK Research and Innovation (UKRI) and the UK Foreign, Commonwealth and Development Office (FCDO) under the MRC/FCDO Concordat agreement and is also part of the EDCTP2 programme supported by the European Union. This project is part of the EDCTP2 program supported by the European Union (grant number RIA2020EF-3008-COVAB) Initial specimen collections were supported by the University of Glasgow GCRF COVID-19 Rapid Response Fund (Uganda COVID-19 Serological Responses UGANCOSER).
• Keywords: SARS-CoV-2, spike-protein, Nucleoprotein, RBD, ELISA validation and optimization, antibody seropositivity cut-offs, Uganda, Sub-Saharan Africa.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Cotten, Professor Matthew
• Authors: Oluka, G. K., Namubiru, P., Kato, L., Ankunda, V., Gombe, B., Cotten, M., Musenero, M., Kaleebu, P., Fox, J., and Serwanga, J.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
• Journal Name: Frontiers in Immunology
• Publisher: Frontiers Media
• ISSN: 1664-3224
• ISSN (Online): 1664-3224
• Copyright Holders: Copyright © 2023 Oluka, Namubiru, Kato, Ankunda, Gombe, Cotten, The COVID-19 Immunoprofiling Team, Musenero, Kaleebu, Fox and Serwanga
• First Published: First published in Frontiers in Immunology 14: 1113194
• Publisher Policy: Reproduced under a Creative Commons License