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
Highlights
In early December 2019, a novel zoonotic coronavirus (CoV) caused a cluster of pneumonia cases in Wuhan, China [1]
As an external standard for IgG quantitation, a twofold dilution series starting with 10 μg/ml of angiotensin-converting-enzyme 2 (ACE-2)-Fc was measured in the flow cytometric assay as described above
The novel serological assay we evaluate here exploits 293T cells that express full-length SARS-CoV-2 spike protein in its natural conformation to bind antigen-specific IgM and IgG from patient sera with a subsequent quantification by secondary detection antibodies
Summary
In early December 2019, a novel zoonotic coronavirus (CoV) caused a cluster of pneumonia cases in Wuhan, China [1]. The identification of acutely infected individuals by the detection of viral RNA by real-time PCR [4] was implemented rapidly in the health care of most countries. While this method is highly valuable for the diagnosis of acute COVID-19 cases, specific serological methods are urgently needed to determine seroconversion in general and to characterize the humoral response against SARS-CoV-2. Together with the 2003 SARS-CoV-1 and the 2012 Middle East respiratory syndrome coronavirus (MERS-CoV) epidemic, the SARS-CoV-2 pandemic represents the third betacoronavirus in 20 years that crossed the species barrier and resulted in a significant number of human infections. We describe a novel flow cytometric assay to determine SARS-CoV-2 spike protein-specific antibodies in serum samples. The virus-free assay relies on reagents and devices that are available in many medical and biological research labs and can be adopted in a decentral manner without the need for commercial kits or products that are prone to shortage
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