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. 2020 Sep 9;21(18):6595.
doi: 10.3390/ijms21186595.

Improved Detection of Antibodies against SARS-CoV-2 by Microsphere-Based Antibody Assay

Carol Ho-Yan Fong  1 , Jian-Piao Cai  1 , Thrimendra Kaushika Dissanayake  1 , Lin-Lei Chen  1 , Charlotte Yee-Ki Choi  1 , Lok-Hin Wong  1 , Anthony Chin-Ki Ng  1 , Polly K P Pang  1 , Deborah Tip-Yin Ho  1 , Rosana Wing-Shan Poon  2 , Tom Wai-Hin Chung  2 , Siddharth Sridhar  1 , Kwok-Hung Chan  1 , Jasper Fuk-Woo Chan  1 , Ivan Fan-Ngai Hung  3 , Kwok-Yung Yuen  1 , Kelvin Kai-Wang To  1
Affiliations

Improved Detection of Antibodies against SARS-CoV-2 by Microsphere-Based Antibody Assay

Carol Ho-Yan Fong et al. Int J Mol Sci. .

Abstract

Currently available COVID-19 antibody tests using enzyme immunoassay (EIA) or immunochromatographic assay have variable sensitivity and specificity. Here, we developed and evaluated a novel microsphere-based antibody assay (MBA) for detecting immunoglobulin G (IgG) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein (NP) and spike protein receptor binding domain (RBD). The seropositive cutoff value was set using a cohort of 294 anonymous serum specimens collected in 2018. The specificity was assessed using serum specimens collected from organ donors or influenza patients before 2020. Seropositive rate was determined among COVID-19 patients. Time-to-seropositivity and signal-to-cutoff (S/CO) ratio were compared between MBA and EIA. MBA had a specificity of 100% (93/93; 95% confidence interval (CI), 96-100%) for anti-NP IgG, 98.9% (92/93; 95% CI 94.2-100%) for anti-RBD IgG. The MBA seropositive rate for convalescent COVID-19 patients was 89.8% (35/39) for anti-NP IgG and 79.5% (31/39) for anti-RBD IgG. The time-to-seropositivity was shorter with MBA than EIA. MBA could better differentiate between COVID-19 patients and negative controls with higher S/CO ratio for COVID-19 patients, lower S/CO ratio with negative controls and fewer specimens in the equivocal range. MBA is robust, simple and is suitable for clinical microbiology laboratory for the accurate determination of anti-SARS-CoV-2 antibodies for diagnosis, serosurveillance, and vaccine trials.

Keywords: COVID-19; SARS-CoV-2; antibody assay; flow cytometry; serology.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Determination of optimal microsphere-protein molar ratio for microsphere-based assay. Serum from a COVID-19 patient was used. The mean fluorescent intensity at different microsphere-protein molar ratio are shown for (A) nucleoprotein (NP) and (B) receptor binding domain (RBD), and the corresponding stacked histogram of selected microsphere-protein molar ratio are shown in (C) NP (1:2) and (D) RBD (1:1). Experiment was performed in triplicate with serum specimen collected from 3 different COVID-19 patients and a representative graph is shown. Error bar represents the standard error of mean from 3 replicates.
Figure 2
Figure 2
Dynamic range of microsphere-based antibody assay (MBA) and enzyme immunoassay (EIA) for (A) NP, and (B) RBD. Serial dilutions of COVID-19 serum were used to detect anti-NP IgG and anti-RBD IgG with MBA and EIA. Experiment was performed with n = 3 and a representative graph is shown. Error bar represents the standard error of mean from 3 replicates.
Figure 3
Figure 3
(A,B) Comparison of MFI between 39 COVID-19 patients, 40 influenza patients, and 53 organ donors for (A) NP and (B) RBD. (C,D) Comparison of OD values between COVID-19 patients, influenza patients, and organ donors for (C) NP and (D) RBD.
Figure 4
Figure 4
Cumulative count of seropositive COVID-19 patient specimens detected by MBA and EIA.
Figure 5
Figure 5
Comparison of signal-to-cutoff (S/CO) ratio between MBA and EIA for the serum specimens collected during the convalescent phase of 39 patients. **** p ≤ 0.0001.
See this image and copyright information in PMC

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