Multiplexed flow cytometric approach for detection of anti-SARS-CoV-2 IgG, IgM and IgA using beads covalently coupled to the nucleocapsid protein

Abstract

Flow cytometry has emerged as a promising technique for detection of SARS-CoV-2 antibodies. In this study, we developed an innovative strategy for simultaneous detection of immunoglobulin G (IgG), IgM and IgA. The SARS-CoV-2 nucleocapsid protein was covalently bound to functional beads surface applying sulpho-SMCC chemistry. BUV395 anti-IgG, BB515 anti-IgM, biotinylated anti-IgA1/IgA2 and BV421 streptavidin were used as fluorophore conjugated secondary antibodies. Serum and antibodies reaction conditions were optimized for each antibody isotype detection and a multiplexed detection assay was developed. This new cell-free assay efficiently discriminate COVID-19 negative and positive samples. The simultaneous detection of IgG, IgM and IgA showed a sensitivity of 88·5-96·2% and specificity of 100%. This novel strategy opens a new avenue for flow cytometry-based diagnosis.

Keywords: CBA functional beads; COVID-19; IgG; IgM and IgA antibodies; SARS-CoV-2; flow cytometry; multiplex immunoassay.

Figure 1

Rational strategy overview. (a) Fluorescent polystyrene beads of 7·5 μm reduced by DDT. SARS‐CoV‐2 nucleocapsid protein solubilized in PBS covalently bound to beads surface by sulpho‐SMCC chemistry to originate functionalized beads named as CBA‐N. (b) Schematic representation of the multiplex assay. (c) Gate selection based on bead size. Representative histograms of COVID‐19 positive samples

Figure 2

Serum and antibody dilution. Determination of percentage of positive fluorescent beads (PPFB) by flow cytometry using CBA‐N beads. (a) Serum dilution using anti‐IgG diluted 100‐fold. (b) Anti‐IgG dilution using serum diluted 1000‐fold. (c) Serum dilution using anti‐IgM diluted 100‐fold. (d) Anti‐IgM dilution using serum diluted 1000‐fold. (e) Serum dilution using anti‐IgA diluted 1000‐fold. (f) Anti‐IgA dilution using serum diluted 1000‐fold. A mix of three samples were used. Data are representative of at least two independent experiments and values are expressed in mean ± SD.

Figure 3

Receiver operating characteristic (ROC) curves and Venn diagram. COVID‐19 negative controls (n = 10) and positive (n = 26). ROC curve for (a) IgG. (b) IgM. (c) IgA. AUC, area under de curve; PPFB, percentage of positive fluorescent beads (%); red point, highest Youden index (cutoff). (d) Venn diagram of COVID‐19 positive samples simultaneously compared with the detection of IgG, IgM and IgA. The statistical calculations were performed using MedCalc v.7·12·7.2·0 (MedCal Software bvba)