Application Notes

Reichert SPR Capture Analysis

Appilcation Note #8

Antibody based capture analysis using a Reichert SR7500DC Surface Plasmon Resonance (SPR) system

Non-covalent capture methods are typically used to attach ligands to an SPR surface in instances where the ligand cannot withstand the lower pH needed for covalent coupling or when a ligand needs to be attached in a more oriented manner. This approach is applicable to a variety of types of samples. The example in this application note is where Goat Anti-Mouse IgG is amine coupled to a dextran surface and then used to capture monoclonal Anti-HSA IgG. Antigen binding (HSA) to the Anti-HSA is followed over a series of concentrations and a KD value is determined.

Experimental

The experimental conditions are summarized in the following table:

  • Ligand: Anti-HSA
  • Analyte: HSA
  • Analyte Concentrations: 1.25, 2.5, 5, 10 and 20nM
  • Association Time: 3 min
  • Dissociation Time: 4 min
  • Regeneration: 10 mM Glycine pH 2.0 with 10% Glycerol

Results

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Figure 1: About 2,000 mRIU of Goat Anti-Mouse IgG Fc was amine coupled to the CMD500k dextran surface. For each series of injections, a constant concentration of monoclonal Anti-HSA IgG (50 mg/mL) was captured over the surface, and then varying concentrations of HSA were injected (see table). Both Anti-HSA and HSA were then removed during each regeneration cycle.
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Figure 2: The good reproducibility of the capture step and the chemical stability of the CMD500k surface are seen here. Even after multiple injection regeneration cycles, the surface was stable and gave reproducible results.
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Figure 3: The good reproducibility of replicate injections of HSA can be seen in this figure. HSA was injected at various concentrations (see table) and fit to a 1:1 binding model. The red lines are the fit obtained in Scrubber (Biologic Software). The equilibrium dissociation constant (KD) obtained was 4.93 nM.