Flow cytometry is a powerful multiparametric cell analysis technique that, using fluorochrome-labelled antibodies, allows the simultaneous measurement of multiple physical characteristics of a cell population.
It is based on passing an aligned cell suspension in front of a laser beam. This laser will excite the fluorochromes of the antibodies bound to the cells, causing an emission of intensity proportional to their concentration, which will be picked up by different detectors at different emission wavelengths. In this sense, flow cytometry is a very versatile technique.
Flow cytometry is a powerful method used to measure cell number, cell viability, programmed cell death (apoptosis), cell division, toxicity and differential expression of specific proteins that can help scientists understand the biology of embryonic development, cancer, metabolism and degeneration, disease, drug effects and even aging.
In this sense Immunostep, a leading provider of technologies, tools and services for bioscience research and biopharmaceutical manufacturing, offers a variety of assays, kits and solutions for flow cytometry.
With flow cytometry, it is possible to label one or many molecular characteristics of cell populations in a blood or bone marrow sample (even dissociated tissues), allowing for a more comprehensive, complex, representative and powerful study that complements microscopic morphology analysis.
Multiparametric flow cytometry is gaining in recent years a major role in the area of hematology and immunology, due to the particular characteristics of the technique that allow a substantial improvement in the knowledge of normal haematopoiesis and, as a consequence, a better understanding of the pathophysiology of onomatological diseases and the immune system.
As we have previously explained in previous articles, the SARS-CoV-2 genome encodes 4 structural proteins: the E protein (envelope), the M protein (membrane), the N protein (nucleocapsid) and the S protein (spike protein), where we also find the RBD domain; and SARS-CoV-2 genome also expresses 27 non-structural proteins (which are released after cell infection).
Serological tests available so far can target the different immunoglobulins (isotypes) but they detect them individually, however a flow cytometry assay is able to detect these three isotypes simultaneously.
Why is this interesting? Each of those antibodies give us different information since they are produced by the immune system in different moments within the infection period.
► Immunoglobulin A (IgA) is the first to appear, since it is localized on mucosal surfaces such as the respiratory tract, which is the main route of entry for the virus, although it is also detected in the patient’s serum.
► IgM (immunoglobulin M) also appears in early stages of infection and indicates that the individual is on the initial response to the disease.
► IgG (immunoglobulin G) is produced at a more advanced stage of infection and can persist over time, indicating that an individual has suffered from the disease in the past.
Our investigations in this field have demonstrated that, in the specific case of the SARS-CoV-2 virus, immunoglobulin A is synthesized before than IgM.
In fact, in all our serological test (as we also developed ELISA tests) we have been able to detect IgA before IgM. Furthermore, we have been able to detect in some patients the IgA immunoglobulins even before the onset of symptoms and in this sense, we have observed that specificity values for IgA are higher than for IgM which is the reason why our serological kits are developed against IgG and IgA.
As the pandemic progresses, new manifestations of the virus are discovered, and it is necessary to evaluate more deeply, qualitatively, and quantitatively the immune response to the virus, which involves studying different antibodies in response to different proteins, the number of antibodies, and their duration for different profiles of individuals.
If we use the current available serological assays (ELISAS, or CLIAS) in order to obtain all this information about the immunoglobulins and proteins involved, we would need to spend plenty of reagents and time to evaluate a large number of results.
In this sense, seeking for an alternative to those assays, we developed this multiplex flow cytometry assay, which is highly specific and sensitive measuring the present or absence of 3 types of immunoglobulins against 4 different SARS-CoV-2 viral proteins, simultaneously. These 4 antigens are:
► Receptor-binding domain (RBD) of glycoprotein S. This RBD domain is a stabilized trimer that allows the precise detection of neutralizing IgG antibodies.
► SPIKE protein. We have a trimeric Spike glycoprotein which is the stabilized native form of the Spike protein of SARS-CoV-2. From a diagnostic point of view, the use of trimeric structures for SPIKE and RBD will allow us to detect a wider range of neutralizing antibodies, improving the sensitivity and precision monitoring the immune system response progress.
► Nucleocapsid protein (N). This protein is highly immunogenic and is the most abundant of the virus. It has a high affinity for the viral RNA with which it interacts.
► The main protease of the virus (3CLpro, Mpro) which is only expressed if the virus enters our cells, and it is just as immunogenic as the nucleocapsid. Spanish Researchers demonstrated its huge potential in serology testing.
The key reason we found useful to analyse 4 antigens simultaneously is the heterogeneity of the immune response which variate from individual to individual. The more information we obtain in the analysis of each patient, the better diagnosis we can offer. In addition, by targeting 4 antigens, it is more difficult not detecting false negative from a possible infected or vaccinated person.
► First of all, we have beads coated with the different proteins: These beads are magnetic microspheres of 6 microns size, auto-fluorescent for the FL3 and FL4 channel and are internally dyed with varying intensities of fluorescence. Each bead will be coated with a specific antigen described before.
► When we add the sample, the antibodies present in that sample will bind to the magnetic bead. These antibodies are conjugated to three different fluorochromes: FITC, PE and PE-Cyanine7 respectively.
► Thus, when the solution is added to the flow cytometer, these fluorochromes will emit a signal of the corresponding colour indicating the presence of antibodies of each type for each protein that have bound to the bead.
Furthermore, these beads are supplied at a concentration of 1,000 beads per test. Each of these microspheres would correspond to one test run in an ELISA.
This means that for each test we are performing, it is as if we were performing 1000 ELISAs simultaneously. The result obtained in each test is the average of these 1000 ELISAs, which makes it a very powerful, reliable, and reproducible assay.
The method is easy. The procedure has 8 steps, and it is performed in an hour and a half. After that time, we will have the samples ready to be analyzed in the cytometer. The assay can be performed in any conventional standard cytometer (it is open format) and you do not need a specific software to analyze the results.
Thus, using magnetic beads will allow us to decant more than one tube simultaneously which makes the protocol faster to perform. An important aspect to consider is how to collect the sample, plasma, or serum. Samples must be properly inactivated. For this inactivation, you can place the sample in a heat bath for at least 30 minutes at 56ºC.
The amount of sample to be used is really small, since this assay can be performed with just 5ul of sample.
These tests offer infinite possibilities, and can analyze an enormous amount of information, as we have show in previous articles about some of these research applications. For example, to carry out the sensitivity of the assay, positive and negative (pre-pandemic) samples were analysed on the flow cytometer. In this analysis we could observe a large channel difference of fluorescence between the positive and negative samples with respect to the positive and negative controls analysed. This channel difference can be clearly seen for all 3 Ig so we can confirm that our kit detects positive cases.
On the other hand, for the evaluation of diagnostic sensitivity, a study was carried out in 2 different clinical laboratories in Spain, where samples were analysed and grouped according to the information available for each sample, in relation to the days that had passed since the PCR confirmation and the onset of symptoms.
The pattern obtained for the 3 Ig allows a clear differentiation between positive and negative samples.
In this sensitivity test, we also compared 35 symptomatic samples against negative and/or pre-pandemic samples without SARS-CoV-2 related symptoms, and many other questions and studies were performed in order to evaluate the quality of the test.
If you are interested in having more information about this, you can watch again our full webinar about serology testing and flow cytometry, where we explained these studies and much more, or you can also contact us for further information.
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