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 antigens and to characterise immunity to the disease in order to address key knowledge gaps in the context of 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 to detect 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 the performance of the assays using 128 plasmas obtained before the COVID-19 pandemic (negative controls) and 115 plasmas from individuals with SARS-CoV-2 diagnosis (positive controls), of whom 8 were asymptomatic, 58 had mild symptoms and 49 were hospitalized. Pre-existing IgG antibodies recognizing N, M and S2 proteins were detected in negative controls suggestive of cross-reactive to common cold coronaviruses. The best performing antibody isotype/antigen signatures had specificities of 100% and sensitivities of 94.94% at ≥14 days since the onset of symptoms and 96.08% at ≥21 days since the onset of symptoms, with AUC of 0.992 and 0.999, respectively. Combining multiple antibody 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 required for vaccine development to combat pandemics like the COVID-19.

Evaluating SARS-CoV-2 spike and nucleocapsid proteins as targets for IgG antibody detection in severe and mild COVID-19 cases using a Luminex bead-based assay

“…This could be relevant in the light of a weaker immune response in mild/asymptomatic cases, suggesting that NP antibody levels might be easier to pick-up during serosurveillance studies (higher sensitivity) 21 , especially when overall antibody titers would decrease over time 27 . Consequently, a combination of NP with RBD/S1S2 is also put forward in two independent Luminex bead-based assays that were recently published 12,13 .…”

“…Nevertheless, many commercial ELISAs became rapidly CE-labelled and are currently used for serosurveillance studies 7 . Since ELISAs as well as VNTs have important limitations for large-scale serosurveillance, microsphere bead-based assays using the Xmap Luminex technology have been increasingly developped [12][13][14] . This high-throughput platform allows the simultaneous detection of antibodies against different antigens from SARS-CoV-2, which can significantly increase the specificity of serological testing, in contrast to ELISA that usually includes only one antigen.…”

A comparison of non-magnetic and magnetic beads for measuring IgG antibodies againstP. vivaxantigens in a multiplexed bead-based assay using Luminex®technology (Bio-Plex®200 or MAGPIX®)

“…Non-magnetic beads: A$640/ml/1.25x10 7 beads 20% less than magnetic beads Up to 100 beads available Magnetic beads: ~A$800/ml/1.25x10 7 beads Up to 50 beads available Better beads retention 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 xMAP ® technology has been used to measure antibody responses against other infectious pathogens, such as HIV and influenza [17,18], to a variety of vaccine antigens such as tetanus toxoid [19], and more recently to SARS-CoV-2 [6][7][8]. was not tested in this experiment.…”

“…This study used a panel of 19 different P. vivax proteins and plasma samples from P. vivax-endemic areas to detect P. vivax-specific IgG responses, however the large number of proteins assessed and consistent results obtained, suggest these findings should be generalizable for optimization of the multiplexed bead-based assay for other pathogens. This is important in the context of the ongoing SARS-CoV-2 pandemic, as multiple laboratory assays based on Luminex technology are under development [6][7][8].…”

From multiplex serology to serolomics – a novel approach to the antibody response against the SARS-CoV-2 proteome

“…Thus far, only a few studies have employed either microarray or fluorescent-bead based technologies to develop multiplex SARS-CoV-2 serological assays (Becker et al, 2020;Dobaño et al, 2020;Jiang et al, 2020;Roxhed et al, 2020;Zamecnik et al, 2020;Zhang et al, 2020), all providing high specificity and sensitivity in detecting SARS-CoV-2 antibodies by varying combinations of proteins N and S, as well as subdomains or peptides thereof. Microarraybased studies utilized peptides or proteins of the SARS-CoV-2 proteome (Jiang et al, 2020;Zamecnik et al, 2020;Zhang et al, 2020) allowing for assessment of the immunogenicity of proteins other than N and S. In contrast to fluorescent-bead based technologies, microarraybased assays are, however, not suited for high-throughput analyses of large sample sets.…”