Surface Plasmon Resonance Assay Services

SPR is a highly sensitive technique for accurate measurement of the interactions of two biomolecules with respect to binding kinetics and affinity as well as binding specificity.

Reaction Biology is equipped with two state-of-the-art Biacore 8K instruments with high-throughput capability and extraordinary detection sensitivity. We perform high-throughput fragment library screening, kinetics and affinity determination, binding specificity profiling and antibody characterization, etc.

  • Suitable to advance any analyte including fragments, antibodies, peptides, nucleic acids against any target class including enzymes and non-active proteins
  • SARS-CoV-2 S protein and ACE2 receptor binding assay is available here
  • Deliverables: association rate constant kon, dissociation rate constant koff, binding affinity KD

 

SPR Principle

SPR is an optical method measuring changes in the mass of biomolecules immobilized on a thin metal film. Upon binding an analyte, the refractive index of the metal film changes, resulting in a changed reflection angle of light (the surface plasmon resonance phenomenon).

surface plasmon resonance (SPR) principle light beam reflects on glass prism and shift in reflection angle is produced by molecules binding to the surface

List of Targets

Target HGNC Symbol Synonyms Data
ACE2 ACE2 Angiotensin I converting enzyme 2
BRD4-1 BRD4 Bromodomain containing 4, domain 1 Data sheet
BRD4-2 BRD4 Bromodomain containing 4, domain 2 Data sheet
cGAS (157-522) CGAS Cyclic GMP-AMP synthase, fragment Data sheet
HEMK2 N6AMT1 N-6 adenine-specific DNA methyltransferase 1
HIF1a, 2a HIF1A, EPAS1 Hypoxia-inducible factor 1-alpha, Endothelial PAS domain-containing protein 1
NSD2-SET NSD2 SET domain of NSD2 Data sheet
PRMT5/MEP50 PRMT5/ WDR77 complex Protein arginine methyltransferase 5, WD repeat domain 77 Data sheet
SARS-CoV-2 S protein - ACE2 interaction n.a. Spike glycoprotein, receptor binding domain; Angiotensin I converting enzyme 2
SOS1 SOS1 SOS Ras/Rac guanine nucleotide exchange factor 1, son of sevenless homolog 1 Data sheet

Additional Information

Kinetics analysis

Surface plasmon resonance kinetic chart

Kinetic profile of an analyte-target binding reaction.

SPR detects changes in the refractive index at the surface of a sensor chip as a result of molecular mass changes of a target upon binding of the analyte. The target is immobilized to the surface of the sensor chip. During the association phase, the analyte flows over the surface, and binding to the target is monitored. The flow then switches to the running buffer, and the dissociation of the analyte from the target is monitored.

Example: Co-factor anaylsis

surface plasmon resonance co-factor analysis on substrate competitive inhibitors that bind to its target enzymes

Example of a co-factor analysis by SPR.

EPZ015666 is a substrate competitive inhibitor that binds to its target enzyme, PRMT5/MEP50, only in the presence of SAM or SAM analogs such as MTA and SAH. No dose-dependent responses were observed for analyte binding to apoprotein (left figure). The binding to the MTA-bound target is relatively weak (KD~20µM) with fast kinetics (on/off). The binding affinity increased by ~10-fold for the SAH-bound target (KD~2µM). While the on-rates are similar for the MTA- and SAH-bound conditions, the off-rates are approximately 100X slower. The highest affinity (KD~3nM) and slowest off-rate (100x less than SAH-bound) was observed for analyte binding to the SAM-bound target. Single-cycle kinetics, that do not require a return to baseline in between doses, was used due to the slow off-rate observed for this condition. A slower off-rate indicates longer occupancy of the analyte on the target.
The analyte was tested with 7 concentrations depicted in different colors. 

Example: Binding affinity

surface plasmon resonance compound screening for binding affinity determination

Example of binding affinity determination of an inhibitor to two bromodomains.

The BD1 or BD2 domain of BRD4 were immobilized to the sensor chip surface. The analyte was applied to the chip surface in increasing concentrations approaching saturation.

Right graph: The relative response at equilibrium for each dose was plotted against the analyte concentration to determine the equilibrium dissociation constant, KD. The analyte is 10-times more selective for BD2 than for BD1.

Screening Details

Instruments: Reaction Biology is equipped with two state-of-the-art Biacore 8K units allowing high-throughput screening with 8 channels (4600 compounds per day) with high sensitivity.

Turnaround time: We strive for a 2 week development time for assays with a new target for screening and 2 weeks for the screening with an established assay. Projects are performed on a first-come-first-serve basis.

Screening location: The assay is performed in Malvern, PA, USA.

Sample requirements: The target can be provided by the client, be one of our off-the-shelf proteins, or be custom-made to the requirements of the client and assay. A purity of 90% or more is recommended.
Please see our FAQ for shipping instructions.

Assay Development

Example SPR workflow of a customized analyte-target binding study:

  • Sensor chip preparation: Attaching the target to the sensorchip
  • Assay validation: Testing the behavior of the target on the sensor chip
  • Assay reproducibility: Testing if the optimized conditions are reproducible
  • Protein activity and stability on the sensor chip
  • Analyte testing
    • The binding of the test analyte(s) is measured
    • For kinetics/affinity determination either a 10 concentration multi-cycle or 5 concentration single-cycle measurement will be used (dependent upon the off-rates and regeneration conditions)
    • For screening studies, a single concentration measurement can be used for analyte ranking

Application

SPR – a versatile tool to address many challenges

Target is not an enzyme or unknown substrate

SPR measures the direct binding between analyte and target which is why the target does not need to be an enzyme and no substrate is needed.

Co-factor/competition studies

The impact of various co-factors on analyte-target interaction can be tested.

Fragment-based screening

Low molecular mass fragment compounds (100–300 Da) tend to demonstrate low binding affinity; thus, the compounds require screening at a high concentration. This is better tolerated in the SPR platform than in many biochemical assays.

Antibody screening/
characterization

SPR can be used for antibody affinity determination, determination of kinetic parameters, epitope mapping, binding specificity, and cross-reactivity.

SAR studies

The kinetics of drug binding and unbinding, especially the residence time, play a crucial role in a drug’s in vivo efficacy.  SPR can rank the kinetic selectivity of drug analogs for the selection of the best drug candidates. 

High-information content

Combining kinetic information with affinity and potency data early in the drug discovery process ensures that promising compounds are not being discarded.

Elimination of promiscuous binders

Promiscuous binders, which appear as false positives in biochemical inhibitor assays, can be identified by SPR when used as the secondary screening technology.

Plasma protein binding

SPR-based assays are sensitive high-throughout options to accurately measure plasma protein binding of analytes.