News | White Papers/Newsletters

Reichert Surface Plasmon Resonance used for kinetic analysis and epitope mapping of monoclonal antibodies

Salmonella Typhimurium (S. Typhimurium) is the most common cause of foodborne illness worldwide but can be difficult to characterize. Researchers at Tennessee State and Auburn Universities have addressed this issue in their 2019 article published in the journal Antibodies. Both Surface Plasmon Resonance (SPR) and enzyme-linked immunosorbent assays (ELISA) have been used for this type of analysis, but the sensitivity and reliability of the assays have been problematic. These researchers are working to improve this situation by characterizing monoclonal antibodies (Mabs) used in these assays more thoroughly. They employed a Reichert SR7500DC to characterize S. Typhimurium interactions with various antibodies.1

Looking more closely at the S. Typhimurium bacterium, one finds it has 6–10 flagella with 3 substructures. One of the substructures, the filament, extends into the extracellular space and is comprised of a single species of protein called flagellin. Further, S. Typhimurium has two non-allelic genes that encode two antigenically distinct flagellins, and monoclonal antibodies can bind to one or the other depending on the specificity of the antibody. The development of numerous immunoassays (ELISA and SPR) based on flagellin, including the current SPR research outlined here, have been made straightforward because: (a) a number of different anti-flagellin antibodies are available, and (b) that Salmonella flagellin can be extracted fairly easily.1

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NiHC: High affinity poly-Ni-NTA sensor chips for fragment based drug discovery

Fragment-based drug discovery (FBDD) has become a preferred alternative to high-throughput screening (HTS) to improve the discovery of small-molecule drug candidates. Screening of low-molecular-weight fragments can identify hit compounds with better efficiency and physiological profiles than HTS2.

SPR biosensor technology is one of the primary biophysical methods to screen fragment libraries3 as newer instruments achieve sufficiently high signal-to-noise ratios to generate reliable data despite the low molecular weight and low affinity of many analytes.

In previous approaches to establish FBDD assays using SPR, the ligand was covalently immobilized on the sensor surface with high immobilization levels to ensure that the protein bound stably to the sensor chip surface. Alternatively, biotinylated proteins were immobilized on streptavidin coated sensor surfaces with the inherent drawback that the analyte could non-specifically interact with the streptavidin. Both immobilization methods lack the possibility to remove the bound ligand from the sensor surface which is critical, for example, when working with GPCRs or other sensitive proteins which often denature over long screening campaigns.

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Sensors and surfaces

With Surface Plasmon Resonance (SPR) and diverse surface chemistries, scientists have discovered an invaluable tool with which to investigate molecular and cellular reactions in fields including immunology, molecular biology, cell biology, biochemistry, and many others.

The quality of the data obtained with this technology depends mainly on the sensor chip surface, where the biomolecular interaction takes place, and is transduced into an optoelectronic signal.

For twenty years, XanTec bioanalytics has provided laboratories worldwide with the ultimate in SPR biosensor chips with superior coating technologies. As the biochemistry is extremely versatile, a large choice of topcoats is available to specifically address different experimental needs. Previous innovations from XanTec, which have eliminated several drawbacks of sensor surfaces currently on the market, are now being utilized extensively by corporate and academic users on a variety of SPR instruments. These include hydrogel-based sensor surfaces coated with the linear polycarboxylate HC that significantly improve diffusion characteristics and inhibit the non-specific interactions which occur with carboxymethyl dextran (CMD)-based sensor surfaces. Also included in this HC group are ultra-stable poly-NTA (NIHC) surfaces. Until now, the use of CMD-based NTA surfaces was limited due to their high leaching – this does not occur with the new NIHC chips.

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Innovation in action: University of Pittsburgh chooses Reichert SPR Systems twice

Thomas E. Smithgall, PhD uses Reichert SPR to research therapeutic inhibitors for treatments of cancer and infectious diseases.

Dr. Thomas Smithgall, Professor and Chair of the Microbiology and Molecular Genetics Department at the University of Pittsburgh, is performing research focused on characterizing new drug targets as potential therapeutic inhibitors to treat cancer and infectious diseases such as HIV. A target that has become of high interest to Smithgall's lab is HIV-1 Nef, a small membrane-associated protein that is critical for HIV-1 replication in vivo, immune escape of infected cells, and AIDS progression. Existing antiretroviral therapy does not remove the HIV virus from the body and requires life-long administration to prevent relapse. In addition, cumulative antiretroviral drug exposure could lead to clinical metabolic disturbances and organ damage. To address these issues, the Smithgall lab is focusing on the development of new classes of compounds, particularly small molecules that interfere with the functions of HIV-1.

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Why you should work »Label-free«!

Label-free study of molecular binding interactions using SPR (Surface Plasmon Resonance) provides scientists with several advantages not available via traditional investigative methods like ELISA. This cost-effective SPR technology offers detailed insights into molecular interactions, and eliminates the confounding variables that sometimes result from the use of fluorescent and radioactive markers.

Fluorescent reagents (such as antibodies) can cost from $50 to over $500 per vial. Radioisotopes often require extensive biohazard training. Using multiple fluorescent reagents requires compensation for spectral overlap, which can give either false positive or false negative responses. Invalid outcomes therefore jeopardize the accuracy of the data interpretation and the conclusions of the study. Label-free capabilities simplify experimental protocols and provide more reproducible results. Accurate results facilitate efficient use of financial and human resources.

Perhaps one of the most important advantages of SPR technology is that it increases the integrity of data by minimizing researcher bias. Bias is sometimes involuntarily introduced into studies by using markers which require additional post-analysis and/or are different between labs.

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Kinetics – or the importance of rate constants in biological and pharmaceutical research

When investigating molecular interactions, equilibrium dissociation constants (KD) are commonly used as a measure of the affinity of two molecules for each other. In other words, the KD is a direct measure of the strength of an interaction. Determining the KD is very important to evaluate the biological relevance of an interaction, such as in the study of fusion proteins, DNA-protein binding and for standard protein analysis.

Fig. 1: Sensorgram of a typical surface plasmon resonance (SPR) measurement.
One of the interactants is immobilized on the sensor surface, while the others are free in solution and pass over the surface. A typical cycle of measurement contains association, steady-state and dissociation phases, eventually followed by regeneration; these phases are displayed in the sensorgram.

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Comparison of Biomolecular Interaction Techniques

Surface Plasmon Resonance (SPR)

Surface Plasmon Resonance (SPR) spectroscopy, a technology developed 25 years ago, is now the gold standard technique for the study of biomolecular interactions for a wide variety of analytes – from small molecules in drug discovery to peptides, proteins, viruses and even nano-particles. SPR is a label-free method capable of measuring real-time quantitative binding affinities, kinetics and thermodynamic parameters of interacting molecules. SPR provides the highest quality data, with moderately high throughput, while consuming relatively small quantities of sample.

The information-rich content of an interaction, which is generated in real-time as well as the high sensitivity and accuracy of this method, make SPR an invaluable tool for biological and pharmaceutical R&D, production and QC. SPR is the only technique from those compared that fulfils the requirements of regulatory authorities (FDA, EMA, ICH).1, 2, 3, 4, 5, 6, 7

With Reichert´s robust and flexible SPR systems it is possible not only to perform standard biomolecular interaction analysis, but also to analyze crude samples and undiluted serum. In addition, Reichert's SPR system can also be used in tandem with liquid chromatography systems and/or mass spectrometers.

As a complement to a robust and flexible instrument hardware that requires little maintenance, XanTec bioanalytics, as the leading manufacturer of biosensor chips, offers the broadest range of sensor chips in the marketplace, meeting virtually every experimental requirement.

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