Here, we describe a serological enzyme-linked immunosorbent assay for the screening and identification of human SARS-CoV-2 seroconverters. This assay does not require the manipulation of infectious viruses, can be adjusted to detect different types of antibodies in serum and plasma and is susceptible to desquamation.
Serological assays are vitally important to help define prior exposure to SARS-CoV-2 in populations, identify highly reactive human donors for convalescent plasma therapy and investigate correlates of protection.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a member of the subgenus Sarbecovirus, has spread globally and caused a pandemic with, so far, 3.6 million infections and 250,000 deaths (up to 5 May 2020).
Nucleic acid tests that detect the SARS-CoV-2 RNA genome are now widely used to diagnose coronavirus disease 2019 (COVID-19). However, there remains a great need for assays that measure antibody responses and determine seroconversion.
Although these serological tests are not suitable for detecting acute infections, they support a number of highly relevant applications. First, serological tests allow us to study the immune response to SARS-CoV-2 qualitatively and quantitatively.
Second, serological surveys are needed to determine the precise rate of infection in an affected area, which is an essential variable in accurately determining the death rate from infection. Third, serological assays will allow the identification of individuals who generate strong antibody responses and who can serve as donors for the generation of convalescent serum/plasma therapies. Finally, serological assays can help inform studies that aim to identify antibody responses that correlate with protection from SARS-CoV-2.
Sarbecoviruses express a large glycoprotein (approximately 140 kDa) called the spike protein (S, a homotrimer), which mediates binding to host cells through interactions with human receptors angiotensin-converting enzyme 2 (ACE2) 1, 2.3. Protein S is highly immunogenic and the receptor-binding domain (RBD) is the target of many neutralizing antibodies4. Individuals infected with coronavirus usually mount neutralizing antibodies5 and a neutralizing response to SARS-CoV-2 has been demonstrated in an individual case from day 9 onwards6.
For human coronaviruses, these responses have been linked to protection over a period of time and future studies will show whether there is also a correlation between neutralizing antibodies and protection against SARS-CoV-25 infection. Serum neutralization can be measured using replication-capable viruses, but the process requires several days and must be performed in a biosafety level 3 laboratory to contain SARS-CoV-2. Pseudotyped viral particle-based entry assays using lentiviruses or vesicular stomatitis virus could potentially be used, but these reagents are not trivial to produce.
A simple solution is to use a binding assay, e.g. Eg an enzyme-linked immunosorbent assay (ELISA), with recombinant antigen as a substrate, especially if the ELISA results correlate with the neutralization assay results. Here we report on the development of such an assay and provide a protocol for both the production of recombinant antigens and the ELISA7 methodology.
We generated two different versions of the SARS-CoV-2 spike protein, based on the genomic sequence of the first virus isolate, Wuhan-Hu-1 (ref. 8). The first construct encodes a full-length, trimeric, and stabilized version of the spike protein, while the second produces only the much smaller RBD. Sequences were codon-optimized for mammalian cell expression.
The sequence of the full-length spike protein was modified to remove the polybasic cleavage site, which is recognized by furin, and to add a pair of stabilizing mutations. These two modifications were included to improve the stability of the protein according to the published literature2,9.
At amino acid P1213, the sequence was fused to a thrombin cleavage site, a T4 fold sequence for proper trimerization, and a carboxy (C) terminal hexahistidine tag for purification. The sequence was cloned into a pCAGGS vector for expression in mammalian cells and into a modified pFastBac Dual vector for baculovirus generation and expression in c