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Since their invention in the 1950s, immunoassays have become a standard tool for the detection and quantification of antibodies and other biomarkers present in serum or plasma. Immunoassays are frequently used in clinical research, drug discovery and diagnostics, and are based on the detection of antigen-antibody interactions. To operate, however, most immunoassay technologies require one binding partner to be attached to a surface. This constraint can bring significant challenges, specifically when measuring, characterizing and quantifying antigen-antibody interactions in complex backgrounds such as serum. In particular, conventional immunoassay titers do not readily resolve the fundamental parameters of affinity and concentration.
To address these challenges and allow researchers routine access to the fundamental parameters underpinning antibody-antigen binding in clinical samples, Fluidic Analytics has developed a novel microfluidic in-solution immunoassay platform which enables affinity-based antibody profiling directly in serum of COVID-19 patients. By use of this approach, the two key determining factors required for a complete immune response assessment, affinity and concentration, can be rapidly and independently determined.
All antibodies are NOT created equal: Comprehensive profiling of SARS-CoV-2 antibodies
COVID-19 is a respiratory disease caused by the coronavirus SARS-CoV-2 that produces mild symptoms in most individuals. However, dependent on underlying health issues and other factors such as age, gender, or genetic makeup, symptoms can vary dramatically in severity. A complete and accurate immune response assessment of COVID-19 patients or vaccinated individuals based on the two key determining factors, namely antibody concentration and affinity, is therefore key to
By applying affinity-based antibody profiling against SARS-CoV-2 for a full immune response assessment in COVID-19 patients or vaccinated individuals, our in-solution immunoassay technology could support the rapid development of effective vaccines, antibody-based therapeutics and reliable diagnostic antibody tests to ease the burden on the health systems globally.
Accurate solution phase affinity profiling of a SARS-CoV-2 antibody in blood serum
Diagnostic tests to assess the immune status of COVID-19 patients or vaccinated individuals are routinely performed using enzyme-linked immunosorbent assays (ELISA). While this test determines the presence of antibodies against SARS-CoV-2, the reported antibody titre is 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.
What the test cannot readily provide is a distinction between large numbers of weak-binding antibodies and small numbers of strong-binding antibodies. As these differences in antibody affinity are believed to play a significant role in disease severity as well as in the protection window against re-infection, ELISA tests require confirmation by additional tests such as virus neutralisation tests to address these questions.
Measuring antibody affinity in COVID-19 patient samples ideally makes use of undiluted serum to minimize sample handling and avoid compromising test sensitivity. As most conventional technologies for protein interaction measurements, such as Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI), rely on surface immobilization of one of the binding partners, low signal-to-noise ratios and false-positive results (specifically when measuring antibody affinities directly in complex backgrounds such serum) can be a consequence of the surface adsorption of other more abundant background species in solution.
In addition, many of the existing technologies require significant dilution of serum samples prior to measuring antibody affinities thereby jeopardizing detection sensitivity and increasing the risk of handling errors.
Microfluidic Diffusional Sizing (MDS) is an in-solution technology that is ideally suited for accurate affinity profiling of SARS-CoV-2 antibodies as it allows the characterisation of protein-protein interactions directly in serum. It enables the detection of antigen–antibody interactions by measuring changes in the hydrodynamic radius (Rh) of the antigen upon binding to the antibody without surface constraints and in their native environment. As a result, our MDS-based immunoassay allows the detection, characterization and quantification of antibodies directly in minimally diluted serum for an accurate assessment of the immune response against SARS-CoV-2.
To learn about how our MDS-based in-solution immunoassay platform has been applied to accurately detect and characterize binding affinities of antibodies to SARS-CoV-2 virus proteins directly in human serum click here to read the full application note.
Measuring protein interactions in blood –
It’s not as trivial as it sounds
An accurate assessment of the immune response against SARS-CoV-2 is key when determining the interaction mechanisms and immunological effects of virus-neutralizing antibodies, convalescent serum or therapeutic agents during vaccine or drug development. The ability to detect, characterize and quantify the concentration of high-affinity antibodies that confer effective immunity by use of our in-solution immunoassay technology could enable ranking and selection of best performing candidates based on their efficacy in inhibiting the interaction of virus proteins with their host targets. In addition our technology offers the ability to assay inhibition of receptor binding directly in serum for further evaluation of virus-neutralizing antibody activity.
Another crucial factor in the development of effective COVID-19 vaccines, antibody-based therapeutics and reliable diagnostic antibody tests is the understanding of cross-reactivity with less harmful coronaviruses to avoid implications regarding test accuracy, or negative side effects when developing vaccines. Applying affinity-based antibody profiling with use of our in-solution immunoassay platform could therefore allow the correlation of cross-reactivity levels with the immune response for rapid identification and ranking of the most suited vaccine candidates or therapeutic agents. It may also help improve diagnostic antibody tests by reducing potential false-positive results caused by cross-reactive signals.
Neutralizing antibodies – the immune system’s superheroes
SARS-CoV-2 Antibody Profiling User Guide
Comprehensive Profiling of SARS-CoV-2 Antibodies - All antibodies are not created equal
Measuring protein interactions in blood – It’s not as trivial as it sounds
Microfluidic diffusional sizing (MDS) is a fundamentally novel technology that characterizes proteins and protein interactions in solution, under native conditions and on the basis of physical properties that determine function. As these parameters are assessed directly in solution, MDS eliminates complications typically associated with surface-based measurements such as binding artefacts or non-specific protein adsorption, and enables researchers to obtain valuable information about binding targets that cannot be obtained using other technologies.
MDS, the underlying technology of our Fluidity One-W instrument, exploits the properties of liquid laminar flow to measure the rate of protein diffusion under steady-state flow in a microfluidic chip. This approach allows rapid evaluation of affinity and concentration, the two key determining factors required for a complete immune response assessment, directly in complex solutions such as serum.
Watch the video below to understand how the Fluidity One-W Serum works:
COVID-19: Comprehensive Profiling of SARS-CoV-2 Antibodies - All antibodies are not created equal
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