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. 2020 Jun;57(1):e100.
doi: 10.1002/cpmc.100.

SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, and Test Setup

Daniel Stadlbauer  1 , Fatima Amanat  1   2 , Veronika Chromikova  1 , Kaijun Jiang  1 , Shirin Strohmeier  1   3 , Guha Asthagiri Arunkumar  1   2 , Jessica Tan  1   2 , Disha Bhavsar  1 , Christina Capuano  1 , Ericka Kirkpatrick  1   2 , Philip Meade  1   2 , Ruhi Nichalle Brito  1 , Catherine Teo  1 , Meagan McMahon  1 , Viviana Simon  1   4   5 , Florian Krammer  1
Affiliations

SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, and Test Setup

Daniel Stadlbauer et al. Curr Protoc Microbiol. 2020 Jun.

Abstract

In late 2019, cases of atypical pneumonia were detected in China. The etiological agent was quickly identified as a betacoronavirus (named SARS-CoV-2), which has since caused a pandemic. Several methods allowing for the specific detection of viral nucleic acids have been established, but these only allow detection of the virus during a short period of time, generally during acute infection. Serological assays are urgently needed to conduct serosurveys, to understand the antibody responses mounted in response to the virus, and to identify individuals who are potentially immune to re-infection. Here we describe a detailed protocol for expression of antigens derived from the spike protein of SARS-CoV-2 that can serve as a substrate for immunological assays, as well as a two-stage serological enzyme-linked immunosorbent assay (ELISA). These assays can be used for research studies and for testing in clinical laboratories. © 2020 The Authors. Basic Protocol 1: Mammalian cell transfection and protein purification Basic Protocol 2: A two-stage ELISA for high-throughput screening of human serum samples for antibodies binding to the spike protein of SARS-CoV-2.

Keywords: COVID19; ELISA; SARS-CoV-2; protein expression; serological assay.

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Figures

Figure 1
Figure 1
Graphical protocol overview.
Figure 2
Figure 2
Vector map showing the pCAGGS expression vectors. (A) Shows the plasmid map of pCAGGS containing the sequence of the stabilized, soluble spike. The schematic below indicates the signal peptide, receptor binding domain, ectodomain with stabilizing mutations, thrombin cleavage site, T4 trimerization domain, and hexahistidine‐tag. (B) illustrates the pCAGGS vector encoding for the soluble receptor binding domain. The signal peptide, receptor binding domain, and hexahistidine‐tag are indicated.
Figure 3
Figure 3
RBD screening ELISA reference plate layout. The layout in which samples should be prepared in a 96‐well cell culture plate (dilution plate) is shown. Wells designated for positive (+) and negative (−) controls are indicated.
Figure 4
Figure 4
Confirmatory spike ELISA reference plate layout. The sample layout on the ELISA plate is shown, including the serial dilution steps that need to be performed. Wells designated for positive (+) and negative (−) controls are indicated.
See this image and copyright information in PMC

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