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Comprehensive Profiling of SARS-CoV-2 Antibodies

All Antibodies Are NOT Created Equal

In the quest to accurately identify individuals who are seropositive as well as finding the most effective vaccines against the recently emerged coronavirus SARS-CoV-2, it is fundamental to thoroughly characterize the immune response in the course of the infection or after vaccination. In particular, the virus-neutralizing capacity of the immune system is of vital interest, and accurate tests to evaluate the affinity and quantity of neutralizing antibodies (NAbs) in serum samples of COVID-19 patients or vaccinated individuals are key.

Transcript:

"Hello and welcome to this seminar series. My name is Haris Choudhery.

A detailed understanding of the human immune response is vital to diagnosing and treating infectious diseases like COVID-19.

In this webinar we will review why current technologies provide only an incomplete understanding of the immune response against SARS-CoV-2. We’ll then demonstrate how Fluidic Analytics’ novel in-solution immunoassay platform on the Fluidity One-W serum, measures antibody affinity directly in serum to obtain crucial insights necessary for efficient diagnostics, therapeutics and vaccine development.

COVID-19 is a respiratory disease caused by the SARS-CoV-2 virus. While symptoms are mild in most people, individuals with underlying health issues and risk factors such as age, gender, or genetic makeup can experience severe and even fatal outcomes.

With this pandemic gripping the world, scientists are working to understand the immune response against SARS-CoV-2.

An accurate characterization of the immune response therefore is of great importance. In fact, the more we know about the type, timing and efficacy of the immune response the better we can guide scientists, clinicians and biopharma companies as they develop treatments and vaccines against this deadly virus.

This includes understanding the differences in disease severity to potentially stratify patients as well as  assessing the strength and duration of the presumed immunity of patients or vaccinated individuals. We must also evaluate levels of cross-reactivity with other less harmful corona viruses to aid the development of reliable antibody tests.

All these points highlight that an accurate assessment of the immune response against SARS-CoV-2 is crucial to fuel our ability of fighting back against the virus.

The most frequently used tools for the detection and quantification of antibodies in serum remain standard immunoassays. Immunoassays are based on decades old technologies and are routinely employed in clinical research, drug discovery and diagnostics.

With COVID-19, diagnostic tests to assess the immune status of infected or vaccinated individuals are routinely performed using enzyme-linked immunosorbent assays, or ELISAs.

While standard ELISAS determine the presence of antibodies against the virus, the reported antibody titer is simplistic, since it represents a combination of the concentration of antibodies in the blood sample and the strength of the binding interaction (affinity) of the antibody to the corresponding antigen.

Notably, this type of immunoassay cannot distinguish between large numbers of weak-binding antibodies and small numbers of strong-binding antibodies. As antibody affinity is however believed to impact disease severity as well as retained immunity, standard ELISA tests on their own can’t provide a comprehensive assessment of the immune response against SARS-CoV-2.

Simply put, all antibodies produced during an immune response are not created equal. Specifically, higher affinity antibodies may in fact confer higher protection against the virus. Thus, Standard ELISAs yield only a partial understanding of the immune response.  It remains an incomplete profile that can lead to inaccurate conclusions regarding retained immunity and virus-neutralizing capacity.

At Fluidic Analytics, we have responded to this need and have developed a novel in-solution immunoassay platform on our Fluidity One-W Serum instrument that simultaneously, but independently, measures both the concentration and affinity of antibodies.

Another key point to consider is the characterization of antibodies in their natural environment as this is crucial for predicting their behaviour in-vivo.

Ideally, measuring antibody affinity in COVID-19 patient samples should take place in undiluted serum to minimize sample handling and avoid compromising test sensitivity, however, given the high protein content in serum samples, this is not a trivial undertaking.

Most conventional technologies for protein interaction measurements, such as Surface Plasmon Resonance or SPR or Bio-Layer Interferometry, commonly referred to as BLI, rely on surface immobilization of one of the binding partners and low signal-to-noise ratios. This can lead to false-positive results that can be a consequence of the surface adsorption of other more abundant background proteins in solution. To counteract this, these technologies may require significant serum dilution which in turn may compromise test sensitivity.

The Fluidity One-W Serum overcomes these limitations by measuring protein interactions directly in solution and in their native environment. This revolutionary new approach is ready to tackle the challenges of accurately profiling SARS-CoV-2 antibody interactions in minimally diluted serum without surface constraints.

The following video will explain our technology in more detail. 

(After video)

To highlight the advantages of our novel in-solution immunoassay platform, we have characterized an anti-spike S1 antibody by measuring its binding affinity to the receptor binding domain, or RBD, of the SARS-CoV-2 spike protein. We then compared results obtained from measurements in PBS buffer with affinity measurements directly in serum using recombinant proteins as controls.

For these measurements, the antibody was titrated against a constant concentration of RBD. The titration experiment was performed in PBS buffer as shown in the figure on the left, and in minimally diluted serum as shown in the figure on the right.

Depending on the dilution factor of the anti-spike S1 antibody in the serum experiments, the respective serum concentrations ranged from 91 – 97%.

Despite the high concentrations of serum in these samples, the KD value of 8.4 nM determined in serum mirrored the result in PBS buffer which gave a KD value of 9.6 nM.

Using a similar approach we analyzed convalescent serum of a 21-year old white male. This individual had no underlying health issues and presented as asymptomatic. Affinity-based antibody profiling against SARS-CoV-2 revealed an antibody concentration of 18 nM and an antibody affinity of 7 nM as shown on the heat map in this slide.

This result highlights the ability of our platform to detect and comprehensively characterize the immune response against SARS-CoV-2 directly in COVID-19 patient serum.

As demonstrated by our data, the Fluidity One-W serum is ideally suited to support future decisions on therapy selection, development and ultimately patient care as it enables the identification and selection of high affinity antibodies for therapeutics, including convalescent serum.

  • The identification and selection of effective virus-neutralizing antibodies via receptor competition assays.
  • Ranking of vaccine candidates based on their efficacy.
  • Correlation of ELISA data with presumed immunity.
  • and evaluation of cross-reactivity to improve the detection specificity of diagnostic antibody tests.

In summary, as all antibodies are not created equal, our novel in-solution immunoassay platform enables scientists, clinicians and pharma companies to independently determine antibody concentration and affinity for a comprehensive and accurate immune response assessment against SARS-CoV-2. 

Our approach uses in-solution measurements to eliminate the many drawbacks of immobilized assays including non-specific binding and binding artefacts.

Lastly, our assays can be run in native complex backgrounds like minimally diluted serum to better predict in-vivo behavior.

Thank you for listening, and learning more about the importance of a comprehensive immunoprofiling approach in the fight against COVID-19.

If you would like to discuss a demo of the Fluidity-One-W serum in your lab, please contact one of our scientists at welcome@fluidic.com. We are happy to tailor our virtual or in-person demos to your needs.

We hope that you will join us for our next webinar and please check our website for further updates.

Thank you."

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