Rapid response flow cytometric assay for the detection of antibody responses to SARS-CoV-2

Abstract

SARS-CoV-2 has emerged as a previously unknown zoonotic coronavirus that spread worldwide causing a serious pandemic. While reliable nucleic acid-based diagnostic assays were rapidly available, only a limited number of validated serological assays were available in the early phase of the pandemic. Here, we evaluated a novel flow cytometric approach to assess spike-specific antibody responses.HEK 293T cells expressing SARS-CoV-2 spike protein in its natural confirmation on the surface were used to detect specific IgG and IgM antibody responses in patient sera by flow cytometry. A soluble angiotensin-converting-enzyme 2 (ACE-2) variant was developed as external standard to quantify spike-specific antibody responses on different assay platforms. Analyses of 201 pre-COVID-19 sera proved a high assay specificity in comparison to commercially available CLIA and ELISA systems, while also revealing the highest sensitivity in specimens from PCR-confirmed SARS-CoV-2-infected patients. The external standard allowed robust quantification of antibody responses among different assay platforms. In conclusion, our newly established flow cytometric assay allows sensitive and quantitative detection of SARS-CoV-2-specific antibodies, which can be easily adopted in different laboratories and does not rely on external supply of assay kits. The flow cytometric assay also provides a blueprint for rapid development of serological tests to other emerging viral infections.

Keywords: Antibodies; Coronavirus; Flow cytometry; SARS-CoV-2; Serology.

Fig. 1

Representative samples measured in the flow cytometric assay. As described in the “Materials and methods” section, 293T cells expressing SARS-CoV-2S protein and pcDNA3.1-transfected cells (mock) were incubated with COVID-19 patient sera (S1–S3) or with negative control sera (S4–S6). Bound IgM and IgG were detected with secondary detection antibodies. The left plot shows the gating of the target populations considering the co-transfected fluorescent proteins as transfection markers (BFP and dsRed). The right histograms depict IgM and IgG fluorescence signals in each sample for both cell populations, respectively. The mean fluorescence intensity is shown in numbers

Fig. 2

Analysis of SARS-CoV-2-specific IgM and IgG in serum samples from uninfected individuals or COVID-19 patients. The flow cytometric serological assay was performed with samples from the pre-COVID-19 era (n = 82), samples from individuals with known endemic HCoV infection (n = 8 before infection, HCoV (end., pre); n = 13 after infection, HCoV (end., post)), and samples from PCR-positive COVID-19 patients (n = 16). Shown are the ratios of the MFI values for SARS-CoV-2-expressing and mock-transfected cells for IgM (a) and IgG (b). The cut-off is depicted as dotted line and represents a ratio of 3. Shown are individual serum samples and the group mean. MFI, mean fluorescence intensity; end., endemic

Fig. 3

Longitudinal analysis of SARS-CoV-2 seroconversion in two COVID-19 cases. At each depicted date, serum samples were collected from the respective patient (A + B) and analyzed by the flow cytometric assay for IgM and IgG. The cut-off is depicted as dotted line and represents a ratio of the MFI values for SARS-CoV-2-expressing and mock-transfected cells of 3. Arrows indicate values above the cut-off

Fig. 4

Quantification of SARS-CoV-2-specific antibody levels by an external ACE-2-Fc standard. (A) Defined concentrations of the ACE-2-Fc protein were analyzed by the flow cytometric assay in five independent measurements (each symbol representing one measurement). With the help of this ACE-2-Fc standard, the concentration of a standard serum was defined. The standard serum was measured in a dilution series by the flow cytometric assay (B) and the EuroImmun ELISA (C). 4-PL curve fitting (shown with 95% confidence bands) was used to generate a standard curve for the absolute quantification of unknown samples. (D) Seven randomly chosen sera from PCR-confirmed COVID-19 patients were quantified by ELISA and the cytometric assay for SARS-CoV-2-specific IgG. The plot assesses the correlation between those two assays by Spearman’s rank correlation coefficient