Webinar | SARS-CoV-2 Serology Testing by Flow Cytometry.

Differentiating antibodies generated after SARS-CoV-2 natural infection by from antibodies generated post-vaccination by flow cytometry.

Key Information:

  • Date: 10th June 2021
  • Time: 14:00 – 15:00h (Madrid, Spain)
  • Language: English
  • Format: Online Meeting on Zoom – subscribe to the webinar to receive the link.
  • Price: Free

Objetives:

  • Understand how a serological assay by flow cytometry works allowing us to detect 3 immunoglobulins against 4 viral proteins simultaneously.

  • Watch online and live how this serological assay offers the information about how the antibodies were generated: by natural infection or as a result of the vaccination.

  • Round of questions

Questions & Answers

Which antibodies can be identified with this assay?

 This multiplex flow cytometry assay 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 antibodies of the IgG subtype.
  • SPIKE protein. We have a trimeric Spike glycoprotein which is the stabilized native form of the Spike protein of SARS-CoV-2.
  • Using 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 or type 3C protease (3CLpro, Mpro) which is only expressed if the virus enters our cells, and it is just as immunogenic as the nucleocapsid. Researchers from the CSIC 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. Targeting 4 different antigens a false negative is rarer, and we will be able to detect easier naturally infected and vaccinated individuals. 

Regarding the stabilised trimer of the spike glycoprotein… is it a recombinant protein, but is it comparable in its entirety to the complete sequence of the native protein S? Would it retain the quaternary structure and glycosylation?

Yes, the S protein used in this kit is recombinant and can be entirely compared to the complete sequence of the native S protein. Moreover, it retains the entire quaternary structure and glycosylation since it is expressed in a mammalian expression system.

 

Is Mpro protein more specific than Nucleocapsid protein?

Yes, Mpro protein has demonstrated to be more specific than nucleocapsid.

As a final specificity test carried out in the validation of the kit, the possible cross-reactivity of antibodies against other microorganisms that produce similar symptoms to SARS-CoV-2 infection was analysed. Thus, 15 samples characterized as IgG positive for the following microorganisms were selected: MERS-CoV, H. Influenzae, RSV, Influenza A, Influenza B, Parainfluenza, Adenovirus, Enterovirus M. pneumoniae, Legionella, C. pneumoniae. 

Pre-pandemic samples were used as a negative control. There is a clear pattern that the positive samples tested do not show cross-reactivity with any of these diseases that produce similar symptoms to SARS-CoV-2. We could observe that the results obtained for IgM is less specific than for IgA and IgG. Furthermore, of the 4 antigens tested and included in the assay, it is noteworthy that Mpro is more specific than Nucleocapsid. This is probably due to the high homology of nucleocapsid with other coronaviruses. Mpro has the lowest homology among other coronaviruses, making it the ideal marker for SARS-CoV-2 detection.

Have you developed any ELISA assay to detect Mpro protein?

Yes, we developed an ELISA assay to detect IgG and IgA antibodies against the main protease (Mpro/3CLpro) which has demonstrated to be highly immunogenic and specific. All the information about this kit, performance data and protocol can be found out on the product page accessing this link:

Why do you need RBD and S? how do you explain the differences between them that you have observed?

The main difference in the information we can obtain from the identification of antibodies against the RBD and the full Spike protein is that it has been demonstrated that the antibodies generated against this protein are neutralising, however, if we identify the full S protein, we will also identify non-neutralising antibodies.

In fact, during the studies we have carried out so far, we have been able to observe differences related to the type of vaccines, and we hope to be able to give more data on these differences soon.

Who the assay works?

This is a bead-based immunological assay by flow cytometry. First of all, we will have beads coated with the different proteins.

These beads are magnetic microspheres of 6 microns size, autofluorescent for the FL3 and FL4 channel and are internally dyed with varying intensities of fluorescence. 

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 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 well. 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 those 1000 ELISAs, which makes it a very powerful, reliable, and reproducible assay.

 
How long take to perform the assay?

The procedure has 8 steps, and it is performed in hour and a half. In that time, we will have the samples ready to analyse 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 analyse the results.

Furthermore, using magnetic beads will allow us to decant more than one tube simultaneously which makes the procedure faster to perform. You can watch the full protocol on video accesing this link:

Is it the standard calibrated with any international standard?

The SARS-CoV-2 multiplex assay has been calibrated against the WHO First International Standard for anti-SARS-CoV-2 Immunoglobulin (human).

In this regard, it is possible to report quantitatively the concentration of IgG immunoglobulins in International Units (IU/ml) against RBD or in Binding Antibody Units (BAU/ml) against S, N and Mpro. For this purpose, the kit includes a calibrator or concentration standard with which to make a calibration line by serial dilutions for each of the viral antigens used in the test (RBD, S, N, Mpro) in which to interpolate the fluorescence values resulting from the assay of the samples, thus obtaining the concentration in International Units or Binding Antibody Units corresponding to each sample.

Does the kit include positive and negative controls for IgG, IgA, and IgM?

This kit contains positive and negative controls for the 3 immunoglobulins, (IgA, IgG, and IgM). These controls come in two separate vials.

The function of the positive and negative controls included in the kit is to help evaluating the quality of the assay, comparing the results in AU/ml with the data we provide in the certificate of analysis. You can find below an example of a table from the certificate of analysis for a random batch. These data can vary between batches, and we provided it with the acquisition of the kit.

Whenever possible, it is recommended that all assays include the laboratory’s own negative controls in addition to the one supplied with this kit.

How do you set up the positivity limit?

According to the product data sheet for the interpretation of the results, the threshold or borderline value is equal to the mean value from the median fluorescence intensity values of the replicates corresponding to the negative control times 3 times the standard deviation.

Threshold (limiting value) = µ (median) ± 3σ (of the negative control).

You can find all the information about the kit components, execution protocol and interpretation of the results on the product page of the website by accessing the following link.

There is any technical report of the assay?

Yes, we have shared plenty of resources and materials about the assay on the product page, where you can also find the Technical Data Sheet and articles with all the information about the assays carried out to validate the assay.  You can access so this product page with this link:

Have the test been validated with different Flow Cytometry platforms?

Yes. the assay has been validated for the main flow cytometers of well-known commercial brands such as Becton Dickinson, Beckman Coulter and Cytek with optimal results

Are there data about the immune response after vaccination?

Yes, we carried out a study among individuals vaccinated with Pfizer, Moderna, and AstraZeneca vaccines. Comparison of IgG and IgA responses against the four SARS-CoV-2 antigens of 65 vaccinated individuals was compared with that of naturally infected individuals. As expected, vaccinated individuals only developed immunoglobulins against the S and RBD antigens, and IgG was the predominant isotype among these vaccinated individuals. On the other hand, sera from naturally infected donors showed antibodies against all four viral antigens included in this kit.  
Thanks to this test, we can confirm that only antibodies are produced against the protein used in the vaccine, while in a person who has been infected, antibodies are also produced against the other antigens included in this test. The use of this test makes it possible to discriminate between antibodies generated by natural infection and those generated by vaccines. In this sense, Mpro plays a fundamental role since it is considered a non-vaccine antigen, as it is not included in any of these vaccines, not even in those developed with inactivated viruses. In that case, people vaccinated with inactivated viruses will generate antibodies to SPIKE and RBD, but they will also generate antibodies to nucleocapsid. But at no time against Mpro, which will allow us to differentiate whether a person has developed antibodies to the vaccine or to a natural infection.

The bar graphs below compare the results between positive patients (natural infection), negative patients (no previous contact with the virus or pre-pandemic samples), and vaccinated people for each of the Ig and antigens included in the test.
 
In addition, the test will give us information about the vaccinated population, how they are responding and whether they are generating antibodies to these vaccines. In other words, the test will allow us to carry out a vaccine effectivness monitoring role. 

Can this assay predict severe disease?

In order to validate the specificity of the test we carried out a study where we compared 35 symptomatic samples against negative and/or pre-pandemic samples without SARS-CoV-2 related symptoms. We could see that the expression of the 4 antigens tested was quite high compared to samples without symptoms.

Furthermore, the sensitivity of the new method was evaluated by testing for the presence of antibodies against the four SARS-CoV-2 antigens (S, RBD, NP, Mpro) in 44 plasma samples including 29 COVID-19 patients, 14 with mild disease and 15 with severe disease.

Each plasma sample was tested at a range of dilutions for the three Ig isotypes (IgA, IgG, IgM). Initial analysis showed a clear difference between the signal obtained for IgG antibodies against the four antigens between healthy controls and COVID-19 patients. As expected, although SARS-CoV-2 specific IgA and IgM antibodies were detected in several patients, they were not present in all sera tested. By testing all 4 antigens and all 3 Ig simultaneously, we can classify patients according to disease severity.

Did you face special challenges setting-up the cytometer prior to run the assays? – which tips could you provide to avoid issues during sample acquisition?

To the correct configuration of the cytometers, you can check the settings data for different platforms on our product page, as well as a video tutorial on how to run the assay protocol and how to perform the gating strategy in the cytometer. In particular, one recommendation we make in this regard is not to purchase the beads at maximum speed in order to avoid doublets and debris.

Access the following link to download these materials or contact us for additional personalized information.

Regarding the resolution and separation of the population of beads in conventional and non-spectral cytometers, is it possible that electronic noise may hinder the choice of beads or mask results? does the threshold for acquisition need to be low?

The performance of this assay has demonstrated to be optimal for any conventional cytometer, in the product page of the kit we provide the data of the settings for the correct configuration of the cytometer, and we include the treschold to be able to perform the acquisition correctly. As an example, we attach an example of how the 4 populations are separated in a non-spectral cytometer (BD Facs Calibur):

Is there any association between immune response and type of vaccine?

Yes, according to studies we have carried out so far, we have been able to identify some differences in the immune response depending on the type of vaccine, and we could differentiate different types of immune response profiles to vaccines. We hope to be able to continue to expand these data in the future in this regard.

Not all individuals who have passed the infection test positive in antibodies against the nucleocapsid protein so how can we say that a positive nucleocapsid is not due to the vaccine and not to a natural infection?

We have observed during the validation of the assay cases in some paediatric samples where antibodies against nucleocapsid protein are not detected. This protein can also be expressed as a result of vaccination with inactivated virus vaccines.

However, the assay also identifies antibodies against the main protease of the virus (Mpro) which is not expressed by vaccination and it is a key enzyme in the viral replication process of the virus during natural infection, so in these cases, it could be differentiated with high accuracy through the data provided with the identification of this protein.

Can you differentiate an individual who suffered from natural infection and has been also vaccinated from an individual who just passed the infection?

We have been able to observe a differentiation of profiles in this sense when IgM expression is at its highest, given that we have not found cases of high titres of IgM antibodies in vaccinated individuals, the antibodies generated as a result of vaccination are predominantly IgG and IgA against S and RBD proteins.

Have you evaluated the differences in the concentrations of antibodies identified over time?

Yes, we have been able to identify significant differences in the concentrations of antibodies identified over time. We hope to be able to provide more data about this subject in the future.

Event organized within the framework of activities related to IDIAL-NET project which is co-financed by the European Regional Development Fund (ERDF) through the INTERREG V-A Spain-Portugal Program (POCTEP) 2014-2020 of the European Commission.