What is Surface Plasmon Resonance?

Surface Plasmon Resonance (SPR) provides a means for monitoring molecular interactions in real time. In an SPR assay, a molecular target, such as a protein or nucleic acid, is immobilized to the surface of a sensor chip and then a second molecule in solution (drug compound or protein binding partner, for example) is passed over this surface. Any binding interaction results in changes in the refractive index for light reflected on the sensor surface. This change is detected, and the output of the SPR assay is presented in the form of a sensorgram, which is a measure of binding response over time.

What Are the Advantages of an SPR Assay?

With optimized assay conditions the kinetics of ligand binding and the equilibrium dissociation constant (KD) can be established. SPR and BLI are the only biophysical techniques that will determine kinetic association (ka) and dissociation (kd) rates. The kinetics, in particular the residence time (derived from kd) of a drug, is often better correlated with its clinical efficacy and duration of action. For example, drugs with slower off-rates give longer duration of action, which could lead to a once-a-day dosing.

Simple affinity determination is unlikely to inform on the impact of structural changes made in the same chemical series.

  • The specificity of an interaction can be determined, establishing whether the ligand cross reacts with other molecules
  • Competition and co-binding assays can be established for binding site validation and binding site mapping (e.g., active site versus allosteric site)
  • Reagent requirements are low
  • SPR assays have a high degree of sensitivity, flexibility, and throughput

Charles River has an experienced surface plasmon resonance team with knowledge of a variety of assay formats. We are able to work with a wide variety of protein targets, antibodies, and oligonucleotides, and provide full guidance in SPR assay design and data interpretation. We will also provide recommendations and options for reasonable next steps following your assay.

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How is SPR Used in Drug Discovery?

Hit Finding and Initial Affinity Determination

  • Single or two-dose screening activities
  • Fast follow-up multi-dose response studies; analyte titration up to a saturating concentration to generate a binding isotherm
  • Stoichiometry and level of surface activity can be derived from comparison to theoretical maximal response values

Binding Characterization

  • Multi-dose analyte titrations to determine ka, kd, and KD
  • Assay methods can be tailored to the interaction type, with full control over association and dissociation times
  • Measurable kinetic ranges:
    • ka: 102 - 107 M-1 s-1
    • kd: 1-10-6 s-1
  • Multiple cycle types and regeneration methods available, allowing for assay refinement on “slow-off” interactions

Mode of Action (MoA) Studies

  • Assessment and comparison of measured kinetics to differentiate binding mechanisms
  • Competition and co-binding assays for binding site validation (i.e., active site versus allosteric site)
  • Characterization of additional interactions to various ligand-bound states of a target molecule

Surface Plasmon Resonance Application Areas

SPR Assays for Biologics

  • Specificity and cross-reactivity studies
  • FcγR and FcRn binding

SPR Assays for Small Molecules

  • Compound to target binding characterization
  • Fragment library screening
  • Plasma protein binding assays (aiding DMPK)
  • PROTAC binding characterization (binary or tertiary assay)

Frequently Asked Questions (FAQs) About Surface Plasmon Resonance Assays

  • Which SPR instruments are available at Charles River?

    We have two high throughput systems, a Biacore 8K+ and a Bruker SPR-24 Pro. Additionally, we have three Biacore T200 instruments and one Biacore S200 instrument. These are highly sensitive systems suitable for detecting signals from low molecular weight analytes.

  • Do I need to provide protein for SPR studies?

    No. While we are happy to work with reagents provided by clients, there is the option to buy commercially available protein, or we can offer a protein production service.

  • Does a target protein always need to be tagged for SPR experiments?

    Not necessarily; there is a range of immobilization techniques that can be applied for untagged proteins. Please talk to our structural biology team to discuss your specific research requirements.

  • We already have an SPR assay developed. Can we transfer this to Charles River, and will it save on costs?

    Yes, we frequently run surface plasmon resonance (SPR) assays that have been developed externally. This means we would not need to include a method development phase, so costs are reduced. There would, however, be a small assay transfer charge.

  • When would you not run a surface plasmon resonance (SPR) assay?

    If you are expecting your binding interaction to be very weak (KD in the mM range) then SPR is not the best technique to use. Equally, if you have a potent molecule where binding kinetics involve a very slow dissociation rate, it may not be possible to fully characterize this interaction (instrument limit for dissociation rate is around 1 x 10-5 s-1).

    The observed SPR response is dependent upon the number of molecules immobilized and also the ratio of the molecular weights of the immobilized molecule and the molecule in solution. If you have a very large protein on the surface (>150 kDa), then detection of binding of very small molecules in solution will be the most challenging.

    It is not advised to test crude biological samples (such as plasma); you are unlikely to obtain clean data because of the complex mixture of molecules present and there is also the risk of blocking the instrument fluidics. Similarly, if you are working with compounds which are known to have very poor solubility in aqueous buffers and/or prone to aggregation, then this is likely to be limiting.

    Talk to our team to review your research options and define the best approaches to support and accelerate your research. Alternatively, visit our Structural Biology homepage to learn more about the structure-based drug design services we offer.