A recent study published on the preprint server medRxiv* discusses a simple, sensitive, and quantitative serological test, based on fluorescence-activated cell sorting (FACS) assay to evaluate antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, the researchers used flow cytometry in the ‘Jurkat-S&R-flow test’ to detect the antibodies present in the sera/plasma by binding them to the S (spike) protein expressed at the surface of Jurkat cells.
The SARS-CoV-2 S protein is highly immunogenic and plays a key role in the entry of the virus into the host cell. Thus, coronavirus disease 2019 (COVID-19) related serological tests often look for the presence of antibodies against the S protein in individuals infected with SARS-CoV-2.
Study: A simple, sensitive and quantitative FACS-based test for SARS-CoV-2 serology in humans and animals. Image Credit: totojang1977 / Shutterstock.com
*Important notice: medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
Serological tests
Serological tests detect antibodies generated in response to natural infection, vaccination, or both.
Since the presence of antibodies usually correlates with recovery in patients, these tests have not been of much use in a clinical setting. However, such serological tests shed important light on the understanding of the pathophysiology and evolution of SARS-CoV-2 throughout the pandemic.
The most commonly used methods for COVID-19 serodiagnostics are either the enzyme-linked immunosorbent assay (ELISA) or ChemiLuminescent Immunoassays (CLIA). Despite good sensitivity and specificity, these methods present several significant drawbacks including high cost, lack of modularity, and venipuncture requirements by trained personnel.
While ELISA is quantitative, both of these assays are not qualitative and saturate rapidly, thus limiting their dynamic range. Furthermore, both of these assays are designed to detect human antibodies, and thus cannot be used to follow serological responses in animal models.
Because lateral-flow immune assays (LFIA) could be used in a point of care setting on capillary blood obtained by finger pricks, these methods were popular early on in the pandemic. However, the general performance of LFIA is low with poor sensitivity and reliability.
Despite these challenges, serological tools are used across the globe. Using these tools, the seroprevalence in a population and immunity of individuals is evaluated.
Further, seroprevalence serves to address questions regarding the immune protection against the SARS-CoV-2. For example, such information can indicate how long this protection will last, how is it affected by the different vaccines, and how the vaccine-induced protection is affected by a prior SARS-CoV-2 infection.
Jurkat-S&R-flow test
As an alternative to current serological assays, the researchers in this study developed a quantitative serological test based on the S-flow test demonstrated in previous studies. To this end, the researchers looked to detect the S-protein expressed on human cells using flow cytometry.
“The performances had compared favorably with three other serological tests (two ELISA directed towards the S or N (nucleocapsid) proteins, and a luciferase immunoprecipitation system (LIPS) combining both N and S detection).”
Using transduction with a lentiviral vector, the researchers expressed high levels of the SARS-CoV-2 spike protein in Jurkat cells (Jurkat-S) while also expressing the mCherry red fluorescent protein in the second population of Jurkat cells (Jurkat-R) to serve as a negative control.
For the test, the researchers mixed equivalent numbers of the two cell lines and used sera or plasma at a final dilution of 1/100 to label 2.105 cells of this mix. This mixture was then incubated for 30 minutes at room temperature before being placed on ice for an additional 30 minutes. This initial incubation remarkably enhances the staining signals.
Cells were then washed before being incubated with a green fluorescent secondary antibody and washed again for flow cytometry analysis. This preparatory step done together for both the cell lines ensures labeling under exact conditions and avoids any differences rising due to labeling procedures. This labeling step also reduces the number of FACS samples to be processed by a factor of two.
In samples without the antibodies against the S-protein, the signals found on Jurkat-S and Jurkat-R populations are similarly green. With samples containing antibodies against the spike protein present in the plasma or serum were used to label the cells, the result is a marked difference in the green signals detected on the Jurkat-S cells compared to the Jurkat-R cells.
The difference in the fluorescence intensity between these two populations provides a quantitative evaluation of the amounts of antibodies in the serum or plasma used to label the cells.
The numbers shown in red correspond to the relative specific staining. Thus, the researchers defined the following equation for their purposes:
RSS = signal Jurkat-S – signal Jurkat-R / signal negative control
“Since the Jurkat-R&S-flow test calls for the use of both a flow cytometer and cells obtained by tissue culture, it is clearly not destined to be used broadly in a diagnostic context, but its simplicity, modularity, and performances both in terms of sensitivity and quantification capacities should prove very useful for research labs working on characterizing antibody responses directed against SARS-2, both in humans and animal models.”
The researchers compared the performance of the Jurkat-S&R-flow test on clinical samples with the other two other serological tests; namely, the Wantai commercial RBD ELISA test and the hemagglutination-based test. While each of these tests showed similar sensitivities, the ELISA signals saturated very rapidly and thus were less dynamic than when flow cytometry was used.
This study also showed that the Jurkat-S&R-flow test requires only 1 µL of plasma or serum, which is comparable to the 100 µL required for the ELIS. This aspect of their approach makes it possible to perform this assay on small volumes of capillary blood collected by finger prick.
The researchers demonstrated that the Jurkat-S&R-flow test can be used to follow Ig responses in mice, cats, and dogs.
Cases of alloreactivity
The researchers also addressed the possibility of the presence of alloreactive antibodies against the cell line used in the assay. This could possibly occur as a consequence of pregnancy or past history of receiving a blood transfusion or organ transplant.
To this end, the researchers observed that about 30% of samples contain alloreactive antibodies that increase the signal about five-fold than that which was obtained for the negative control. These observations therefore suggested that pregnancy is not a major cause in the origin of the allo-reactions.
Conclusion
In the current study, the researchers demonstrate that the Jurkat-S&R-flow assay is a versatile, flexible, and affordable approach to measure serological responses against the spike protein of SARS-CoV-2 in both humans and animals.
*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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