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

SARS-CoV-2 and SARS-CoV spike-mediated cell-cell fusion differ in the requirements for receptor expression and proteolytic activation

“…The cells were then washed once in a large volume of PBS and post-fixated in 2% PFA in PBS before analysis on an LSRII flow cytometer (BD Biosciences). Data was analyzed using Flowing software (version 2.5) and GraphPad Prism, version 6, for Windows (GraphPad Software).COVID-19 convalescent serum was collected previously (59) in accordance with ethical requirements (ethics committee UK Erlangen, license number AZ. 174_20 B).…”

“…The cells were then washed once in PBS and then incubated in 10% FCS in PBS for 30 min to block non-specific binding. The cells were then incubated in either convalescent serum at 1:1000 dilution or soluble ACE2-Fc fusion protein at 2 ng/μl, both described elsewhere (58), for 1h in 10% FCS in PBS, followed by one wash in a large volume of PBS and then incubation with Alexa647-coupled anti-human secondary antibody (Thermo Fisher Scientific) at 1:200 in 10% FCS in PBS. The RRV gHΔ21-27-Fc fusion protein, which was used as a control protein, was generated from RRV 26-95 gH-Fc (59) by deletion of the codons for amino acid 21-27, which are important for receptor binding (28), and was produced analogous to the gH-Fc protein in Hahn et al 2013 (59).…”

A pair of non-competing neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model

“…To detect antibodies that bind to SARS-CoV-2-S protein, HEK-293T cells were co-transfected with SARS-CoV-2-S DNA or pCG1-SARS-Fra1-S (CoV-1; position 21492 to 25259 Genbank AY291315SARS-CoV) and a GFP reporter plasmid (e.g., pEGFP-C1) using the PEI method as described previously [56]. Briefly, 2x10 7 semi-confluent HEK-293T in 10 ml D1.5+ (DMEM+ 5% glutamine + Pen/Strep + 1.5% FCS) cells were incubated with 2.5 ml of a transfection mix in D0 (DMEM+ 5% glutamine + Pen/Strep) containing 30 µg of pCG1-CoV-2019-S or pCG1-SARS-Fra1-S and 10 µg of pEGFP-C1 plasmids and 180 µl of PEI (1mg/ml).…”

Multiplexed Flow Cytometric Approach for Detection of Anti-SARS-CoV-2 IgG, IgM and IgA Using Beads Covalently Coupled to the Nucleocapsid Protein

“…The application of flow cytometry in COVID-19 has exploited the inverse format to evaluate the humoral antibody response. Lapuente et al (2020) 23 , Priya Anand et al (2020) 25 and Simard et al (2021) 24 used cell lines expressing the SARS-CoV-2 spike protein for the detection of IgG and IgM, IgG and IgG+IgA+IgM pool in human serum samples, respectively. To the best of our knowledge there is only one study employing a cell-free flow cytometric approach to evaluate the humoral antibody response 18 .…”

“…Recent studies exploited flow cytometric to develop assays to detect COVID-19 seroconversion in humans. In three studies, the spike protein was overexpressed on the surface of cells, allowing the detection of antibodies in patient samples using fluorescent secondary anti-antibodies [23][24][25] . In another study, SARS-CoV-2 antigens tagged with biotin were non-covalently bound to beads coated with streptavidin 18 .…”