SPR Assay Development and Experimental Optimization

print

Overview

Assay development and experimental optimization are essential for obtaining high-quality SPR data. Selecting the most suitable design and optimizing the experimental conditions is of utmost importance for maintaining assay accuracy and reproducibility. This is also one of the most time-consuming steps for SPR researchers due to the low throughput of conventional SPR devices.

The ProteOn™ XPR36 protein interaction array system's novel 6 x 6 fluidics afford the researcher the flexibility to run many different types of experiments on a single platform at a higher-throughput than traditional binding assays. This flexibility in assay design allows for streamlining of large-scale sample processing and optimization.

Related Topics: Label-Free Validation and Characterization and Large and Small Molecule Screening by SPR

Assay Development

The unique 6 x 6 interaction array of the ProteOn system can be used to detect binding between up to six targets and six analytes at the same time, greatly enhancing experimental throughput and the versatility of SPR technology for a wide range of applications. An example of this 6 x 1 kinetic screening approach is seen through the investigation of the model system, TEM-BLIP, where 5 mutant TEM1 proteins are immobilized to the sensor chip surface. A single injection of a concentration series of the BLIP wild type analyte yields robust kinetic rate constants that can be used to model future experiments. (Bronner et al. 2006a, Bronner et al. 2009).

 

Fig. 1. Working model depicting the binding domain interactions between TEM1 and BLIP proteins.

TEM1 Mutant ka (M–1 sec–1) kd (sec–1) KD (M)
R243A/S235A 1.61 x 104 4.48 x 10–4 2.78 x 10–8
K234A 2.11 x 104 8.86 x 10–4 4.20 x 10–8
R243A/S130A 1.29 x 104 1.22 x 10–3 9.46 x 10–8
S235/S130A 3.02 x 104 9.11 x 10–4 3.02 x 10–8
E104A 1.66 x 105 7.46 x 10–3 4.49 x 10–8

 

Kinetic constants for the interactions between mutants of TEM1 and wild-type BLIP. The equilibrium dissociation constant, KD, was calculated from kd/ka.

Relevant webinars for assay development:

Kinetic Screening of an scFv Antibody Fragment Library Using the ProteOn XPR36 Interaction Array System
Presented by Olan Dolezal, PhD (CSIRO Molecular and Health Technologies, Australia)
Click to download recorded webinar: https://biorad.box.net/shared/ra1bne7z9c

A Calcium-Dependent Immunocapture Strategy for Enhanced-Throughput SPR
Presented by John Kulman, PhD (Puget Sound Blood Center, USA)
Click to download recorded webinar: https://biorad.box.net/shared/j00jbuzdet

The Study of Small Molecule-Protein Kinase Interactions using Multiplexed SPR
Presented by Tsafrir Bravman, PhD (Bio-Rad Laboratories)
Click to download recorded webinar: https://biorad.box.net/shared/p0hrnbgxaria4lyuqfkz

Enhancing Throughput of the ProteOn Biosensor in Antibody Screening Applications
Presented by Kevin Lindquist (Pfizer Rinat, USA)
Click to download recorded webinar: https://biorad.box.com/s/meykvrgruuff29oqdrod

Experimental Optimization

The novel microfluidics of the ProteOn system uniquely enable researchers to quickly determine ideal experimental conditions for their interaction of interest. XPR™ technology allows the user to investigate up to six conditions of the target followed by a single injection of up to six conditions of the analyte. The 1 x 1 full optimization configuration of the ProteOn enables rapid determination of the surface density of the target, buffer, or regeneration conditions for the experiment. The combination of different varying target conditions interacted with different analyte concentrations results in a large matrix of optimization conditions. Combing through these optimization conditions sequentially can be difficult and time-consuming; however, by using the ProteOn system, a researcher can complete an entire optimization experiment in two injections (Bronner et al. 2006b).

Channel Buffer pH Ligand Density (RU)
1 3.0 610 ± 6
2 3.5 681 ± 6
3 4.0 742 ± 17
4 4.5 727 ± 5
5 5.0 167 ± 4

 

pH dependence of TEM1 immobilization. TEM1 protein was immobilized in ProteOn acetate buffer, pH 3.0–5.0. Ligand density was determined from the average SPR response of the six interaction spots along each ligand channel.

Fig. 2. The dependence of the BLIP-F142A analyte response, TEM1 ligand density, and TEM1 ligand activity (%Rmax) on the pH of the immobilization buffer. Scales adjusted to align the response of each parameter to the same plot.

Webinars addressing experimental optimization using the ProteOn XPR36 system:

An Introduction to the ProteOn XPR36 Surface Plasmon Resonance (SPR) Interaction System
Presented by Tsafrir Bravman, PhD (Bio-Rad Laboratories)
Click to download recorded webinar: https://biorad.box.net/shared/mhfscze1oz

A Calcium-Dependent Immunocapture Strategy for Enhanced-Throughput SPR
Presented by John Kulman, PhD (Puget Sound Blood Center, USA)
Click to download recorded webinar: https://biorad.box.net/shared/j00jbuzdet

References

Bronner V. et al. (2006a). Analysis of multiple protein-protein interactions using the ProteOn™ XPR36 protein interaction array system. Bulletin 5368.
This tech note describes the investigation of the relative contributions of specific protein substructures and residues to the binding interface between TEM1 β-lactamase (TEM1) and the β-lactamase inhibitor protein (BLIP) (Figure 1) (Albeck and Schreiber 1999). TEM1. Mutated TEM1 residues were used to analyze the consequences of mutations on the binding energetics of the protein interface. Central to this analysis was the fast and accurate "one-shot kinetics" capability of the ProteOn XPR36 protein interaction array system.

Bronner V et al. (2006b). Rapid and efficient determination of kinetic rate constants using the ProteOn XPR36 protein interaction array system. Bio-Rad Bulletin 3172.
The tech note employs the ProteOn system to determine the kinetics of the interaction between interleukin-2 (IL2) and anti-IL2 antibody. The experiment was performed using the One-shot Kinetics™ approach to monitor the interaction between multiple concentrations of the analyte IL2 and the ligand anti-IL2 antibody immobilized with multiple conditions in a single analyte injection. The 6 x 6 interaction array was used to generate 36 sensorgrams simultaneously. It not only increased throughput but also provided novel referencing options.

Bronner V et al. (2009). Rapid screening and selection of optimal antibody capturing agents using the ProteOn XPR36 protein interaction array system. Bio-Rad Bulletin 5820.
The tech note describes how the One-shot Kinetics approach was used to rapidly screen the binding of four antibody capturing agents and seven types of antibody targets. The selection of antibody-binding proteins that provide the optimal binding characteristics for the capture of each antibody type was achieved rapidly in the ProteOn system. The One-shot Kinetics approach allows for the analysis of multiple experimental conditions in a single experiment.

Additional references:

  • Balducci C et al. (2010). Synthetic amyloid-beta oligomers impair long-term memory independently of cellular prion protein. Proc Natl Acad Sci USA 107, 2295-300.
  • Di Fede G et al. (2009). A recessive mutation in the APP gene with dominant-negative effect on amyloidogenesis. Science 323, 1473-7.
  • Pedersen MW et al. (2010). Sym004: a novel synergistic anti-epidermal growth factor receptor antibody mixture with superior anticancer efficacy. Cancer Res 2010 70, 588-97.
  • Yousef M (2007). Advances in rapid monoclonal antibody screening. American Biotechnology Laboratory 25, 26–28.

Related Content

 

  General ProteOn Literature

5390 ProteOn — PIA XPR36 Brochure Click to download
5538 ProteOn XPR36 — Analyzing Protein Interaction Array System Featured Article Reprint Click to download
5413 ProteOn XPR36 Hardware — 36 Interactions on a Single Chip: Label-Free, in Real-Time Product Information Sheet Click to download
5627 ProteOn Manager Software Product Information Sheet Click to download
5404 ProteOn Sensor Chips — Application-Specific Surface Chemistries = Optimized Ligand Activty Product Information Sheet Click to download
5410 ProteOn Protocol Development Kits Product Information Sheet Click to download
5409 Protein Interaction Analysis — ProteOn XPR36 System Ordering Information Sheet Click to download

  Large Molecule Protein-Interaction Analysis

3172 ProteOn PIA — Rapid and Efficient Determination of Kinetic Rate Constants using the ProteOn XPR36 Protein Interaction Array System
Click to download
5412 ProteOn App Guide — Antibody Characterization and Development Using the ProteOn XPR36 PIA System Application Guide Product Information Sheet Click to download
5540 ProteOn — PIA Screening, Ranking, and Epitope Mapping of Anti-Human IL-9 Supernatants
Click to download
5368 ProteOn PIA — Analysis of Multiple Protein-Protein Interactions Using the ProteOn XPR Protein Interaction Array System
Click to download
5449 Protein Interaction Analysis Applications of the ProteOn NLC Sensor Chip: Antibody-Antigen, DNA-Protein-Protein Interactions
Click to download
5358 ProteOn PIA — Mechanisms of Protein Binding: Double-Mutant Cycle Analysis Using the ProteOn XPR36 System
Click to download
5820 ProteOn — Rapid Screening and Selection of Optimal Antibody Capturing Agents Using the ProteOn XPR36 Protein Interaction Array System Click to download
5360 ProteOn PIA — Rapid and Detailed Analysis of Muliple Antigen-Antibody Pairs Using the ProteOn XPR36 Protein Interaction Array System
Click to download
5367 ProteOn PIA — Rapid Optimization of Immobilization and Binding Conditions for Kinetic Analysis of Protein-Protein Interactions using the ProteOn XPR36 PI Array System
Click to download

 

  Small Molecule Analysis

5965 Rapid High-Throughput Screening of Protein Kinase Inhibitors Using the ProteOn PIA System Click to download
5797 Protein Interaction — Rapid Assay Development and Optimization for Small Molecule Drug Discovery
Click to download
5679 ProteOn — Applications of the ProteOn GLH Sensor Chip: Interaction Between Proteins and Small Molecules Click to download
5960 High-Throughput Profiling of Kinase Inhibitors Selectivity Using the ProteOn XPR36 Protein Interaction Array System Click to download
5846 Determining the Binding Kinetics of HIV-1 Nucleocapsid Protein to Six Densities of Oligonucleotide Using the ProteOn XPR36 Protein Interaction Array System
Click to download
5822 How to Perform Excluded Volume Correction on the ProteOn XPR36 Protein Interaction System Product Guide Click to download
Ask an Expert

Call us at 1-800-268-0213

Sign Up for Bio-Rad Updates!
Enter your email address below to receive your choice of the latest news, promotions, and more.