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. 2021 Oct 9;4(4):72.
doi: 10.3390/mps4040072.

SARS-CoV-2 Multi-Antigen Serology Assay

Ramin Mazhari  1   2 , Shazia Ruybal-Pesántez  1   2   3 , Fiona Angrisano  3 , Nicholas Kiernan-Walker  1   2 , Stephanie Hyslop  1   2 , Rhea J Longley  1   2 , Caitlin Bourke  1   2 , Catherine Chen  1   2 , Deborah A Williamson  4   5 , Leanne J Robinson  1   2   3 , Ivo Mueller  1   2 , Emily M Eriksson  1   2
Affiliations

SARS-CoV-2 Multi-Antigen Serology Assay

Ramin Mazhari et al. Methods Protoc. .

Abstract

Serology tests are extremely useful for assessing whether a person has been infected with a pathogen. Since the onset of the COVID-19 pandemic, measurement of anti-SARS-CoV-2-specific antibodies has been considered an essential tool in identifying seropositive individuals and thereby understanding the extent of transmission in communities. The Luminex system is a bead-based technology that has the capacity to assess multiple antigens simultaneously using very low sample volumes and is ideal for high-throughput studies. We have adapted this technology to develop a COVID-19 multi-antigen serological assay. This protocol described here carefully outlines recommended steps to optimize and establish this method for COVID-19-specific antibody measurement in plasma and in saliva. However, the protocol can easily be customized and thus the assay is broadly applicable to measure antibodies to other pathogens.

Keywords: COVID-19; Luminex; SARS-CoV-2; serology.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic of serological assay workflow. Created with BioRender.com (accessed on 24 August 2021).
Figure 2
Figure 2
An example of plate layout containing a blank and negative control as well as a standard curve (S1–S10). Remaining wells are used for individual samples.
Figure 3
Figure 3
Standard curves for (a) SARS-CoV-2 antigens and (b) other antigens in human plasma, such as respiratory viruses and human seasonal coronaviruses. Levels of IgG antibodies are presented as log10-transformed median fluorescence intensity (MFI) values.
Figure 4
Figure 4
Reactivity of (a) anti-S1, (b) anti-S2, (c) anti-NP and (d) anti-RBD and anti-Spike human monoclonal antibodies to our 14-antigen panel. Levels of IgG or IgM antibodies are presented as log10-transformed median fluorescence intensity (MFI) values. Only the reactive and/or cross-reactive antigens are highlighted, all other antigens are in grey (including the seasonal coronaviruses, tetanus toxoid and influenza viruses).
Figure 5
Figure 5
Comparison of standard curves in multiplex (red) and single-plex (blue). Levels of IgG antibodies in human plasma are presented as log10-transformed median fluorescence intensity (MFI) values.
Figure 6
Figure 6
Standard curves for (a) SARS-CoV-2 antigens and (b) other antigens, such as respiratory viruses and human seasonal coronaviruses. Levels of IgM antibodies are presented as log10-transformed median fluorescence intensity (MFI) values. Solid triangles depict the negative values for each SARS-CoV-2 antigen, run at a 1:100 dilution.
Figure 7
Figure 7
Standard curves for (a) SARS-CoV-2 antigens and (b) other antigens, such as respiratory viruses and human seasonal coronaviruses in plasma (left panels) and saliva (right panels). Levels of IgA antibodies are presented as log10-transformed median fluorescence intensity (MFI) values.
See this image and copyright information in PMC

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