Highly Sensitive and Specific Multiplex Antibody Assays To Quantify Immunoglobulins M, A, and G against SARS-CoV-2 Antigens

Abstract

Reliable serological tests are required to determine the prevalence of antibodies against SARS-CoV-2 and to characterise immunity to the disease in order to address key knowledge gaps in the COVID-19 pandemic. Quantitative suspension array technology (qSAT) assays based on the xMAP Luminex platform overcome the limitations of rapid diagnostic tests and ELISA with their higher precision, dynamic range, throughput, miniaturization, cost-efficacy and multiplexing capacity. We developed three qSAT assays for IgM, IgA and IgG to a panel of eight SARS-CoV-2 antigens including spike (S), nucleoprotein (N) and membrane (M) protein constructs. The assays were optimized to minimize processing time and maximize signal to noise ratio. We evaluated their performance using 128 pre-pandemic plasmas (negative controls) and 104 plasmas from individuals with SARS-CoV-2 diagnosis (positive controls), of whom 5 were asymptomatic, 51 had mild symptoms and 48 were hospitalized. Pre-existing IgG antibodies recognizing N, M and S proteins were detected in negative controls suggestive of cross-reactive to common cold coronaviruses. The best performing antibody/antigen signatures had specificities of 100% and sensitivities of 95.78% at ≥14 days and 95.65% at ≥21 days since the onset of symptoms, with AUC of 0.977 and 0.999, respectively. Combining multiple markers as assessed by qSAT assays has the highest efficiency, breadth and versatility to accurately detect low-level antibody responses for obtaining reliable data on prevalence of exposure to novel pathogens in a population. Our assays will allow gaining insights into antibody correlates of immunity and their kinetics, required for vaccine development to combat the COVID-19 pandemic.

A flexible, pan-species, multi-antigen platform for the detection and monitoring of SARS-CoV-2-specific antibody responses

“…During the course of this work, many reports of serological diagnostic assays, including those based upon the Luminex format (15)(16)(17)(18)(19)(20)(21)(22)(23), have been presented. Rather than developing an additional diagnostic platform, we emphasize here the utility of the Luminex system as a research tool -for monitoring changes in antibody responses over time, detecting antibody responses in different sample matrices and for rapid adaptation and evaluation of new antigen formulations.…”

Evaluation of Antibody Response in Symptomatic and Asymptomatic COVID-19 Patients and Diagnostic Assessment of New IgM/IgG ELISA Kits

“…Up to date, there is no sufficiently effective antiviral drug to treat COVID-19. Therefore, the development of accurate and reliable diagnostic serological assays that can be readily applicable is crucial [1,2]. These assays should provide guidance to determine the seroprevalence of antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the individual and community levels, identify immune presumptive protected persons who can serve as potential plasma donors, and for vaccine development [1].…”

“…Therefore, the development of accurate and reliable diagnostic serological assays that can be readily applicable is crucial [1,2]. These assays should provide guidance to determine the seroprevalence of antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the individual and community levels, identify immune presumptive protected persons who can serve as potential plasma donors, and for vaccine development [1]. In addition, serology assays are expected to play a critical role in testing the currently approved vaccines' efficiency by measuring the level of produced antibodies, determining their durability, and identifying thresholds of protection [3,4].…”

A comparison of non-magnetic and magnetic beads for measuring IgG antibodies against Plasmodium vivax antigens in a multiplexed bead-based assay using Luminex technology (Bio-Plex 200 or MAGPIX)

“…Whilst these results were obtained in the context of P. vivax-specific IgG responses in individuals from malaria-endemic areas, the large panel of proteins used and consistent results obtained for all proteins suggest these results can be applied to guide studies in other fields. Luminex 1 xMAP 1 technology has been used to measure antibody responses against other infectious pathogens, such as HIV and influenza [14,15], to a variety of vaccine antigens such as tetanus toxoid [16], and more recently to SARS-CoV-2 [17][18][19].…”