Since the coronavirus disease 2019 (COVID-19) pandemic emerged in Wuhan, China, in December 2019, scientists have developed a range of tests to detect the presence of infection with its etiological agent, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
Serology is likely to play a major role in understanding the epidemiology and history of the infection, and in disease surveillance. Researchers in the U.S. - based at the University of Georgia, Emory University, and Texas A&M University - have released a new study that describes a powerful but inexpensive tool designed to detect and monitor specific antibodies to SARS-CoV-2 over time. This could prove a boon to researchers in this field, enabling them to track antibody development to several viral proteins in multiple species and all kinds of sample types.
*Important notice: medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
The findings of their study have been released on the medRxiv* preprint server.
The study aim
The researchers aimed to develop a serologic assay that could detect all antibodies specific to SARS-CoV-2, belonging to various isotypes and reacting to different antigens from multiple species. The reagents used in this assay had to readily available, commercially, or inexpensively produced in a typical biomedical laboratory.
Range of antigens
Most serological assays for this virus in current use depend on the SARS-CoV-2 spike (S) protein and its receptor-binding domain (RBD) fragment for antigen recognition. The researchers first used codon-optimized S, RBD and nucleocapsid (N) antigens in E. coli systems, and compared their detection with that of S and RBD from mammalian cells.
However, the E. coli-generated S and RBD proteins did not bind immunoglobulin G (IgG) antibodies specific to the virus in sera from infected individuals, though the N protein did. Protein fragments such as E. coli-produced versions of the viral open reading frame (ORF) 3b and ORF8 were not efficient detectors of antibodies in one of three human sample cohorts.
The truncated N protein also showed low reactivity. However, deglycosylated recombinant Spike-M protein displayed significantly higher binding to antibodies in most subjects with low or moderate anti-S antibodies.
Luminex platform
Earlier work showed that the Luminex multiplexing system fulfilled these requirements, and delivered precise results from minute amounts of the sample. It also had a wide range of determination. The antigens used can be rapidly produced in E. coli cultures, with protein for over 30,000 assays from one 150 mL culture. Each sample requires less than one microliter of serum, or an eluted blood spot, and can be assessed for a hundred different antigens.
Since SARS-CoV-2 also affects many other species, the ability of the assay to detect IgG antibodies specific to the virus was tested in dogs and cats as well, showing modest-to-high detection.
The researchers also constructed and tested two new epitopes reported to be neutralizing antibody targets, namely, Spike-4P and Spike 403-505. They were found to be specifically recognized by sera from infected individuals but at a different level, in every cohort, from that of the spike-M protein. Longitudinal follow up showed the persistence of these differences in serological response, with the responses occasionally reversing their direction after some months.
The cost for each assay could be $5 at the lowest, varying with the type and number of antigens, and the sources of the reagents used. The plasmids used in the current study were generated in E. coli systems, with antigen generation and conjugated bead production through simple protocols. They are being released through Addgene for wider availability.
The roadblock for most laboratories will be the need for a Luminex assay reader, which can cost up to $30,000 new. This system has been the focus of the attempt to develop a multi-antibody SARS-CoV-2 diagnostic assay because of its usefulness.
Applications of this assay
Not only is it possible to trace the changes in antibody response over time, but antibodies may be detected in different sets of samples. This platform will also allow the rapid adaptation and evaluation of new antigens. The researchers were able to confirm, yet again, the wide range and antigen specificity of SARS-CoV-2 antibodies in various animals and in different individuals, in a large variety of samples, as well as their temporal profile.
The use of a multiplexed assay for SARS-CoV-2, with multiple antigens, is not only useful in the sensitive detection of past infection, but yields deeper data on the immune response to the pathogen in a shorter time and with less of the sample.
The simultaneous use of reference viral antigens rules out artefactual changes in the antibodies to SARS-CoV-2 rather than actual time-related and antigen-associated variations. Such an assay could thus also help to trace the increase in specific antibodies following re-exposure or vaccination.
Importantly, the use of the assay was found to yield specific antibodies reacting to the deglycosylated viral S protein, linked S epitopes and S fragments. The implication is that the E. coli spike was improperly folded, which led to its inability to be captured by specific anti-SARS-CoV-2 antibodies, rather than its lack of specific glycans. The Spike-4P and Spike 403-505 fragments generated in E. coli did not suffer from misfolding issues.
These constructs of the spike protein were the result of epitope discovery reports, and were included on the assay platform in less than 40 days from the first published report on their identification. The researchers comment, “The association of the changing pattern of responses to these and other SARS-CoV-2 epitopes could be of use in identifying response patterns that associate with risk of infection/reinfection, or protection from severe disease.”
The ease of use, the diversity of sample types and applications, and the multiplexed nature of the assays, as well as the ability to include new antigens as required, make this a useful tool in research on novel infectious agents such as SARS-CoV-2, where the dominant epitopes are still in the process of discovery or production.
*Important notice: medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
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