Reichert SPR Concentration AnalysisAppilcation Note #9
Determining the Concentration of an Antigen Sample Using the SR7500DC Dual Channel Surface Plasmon Resonance (SPR) System
The concentration of an analyte can be determined directly using SPR. In this example, anti-human serum albumin mouse monoclonal (Anti-HSA) was immobilized directly to the sensor surface at a high level (greater than 1500 micro RIU). A range of antigen concentrations (human serum albumin or HSA) was injected over the surface and was expected to encompass the concentration in the unknown samples. The high surface concentration of antibody created a diffusion controlled binding response where the initial response was proportional to the concentration of the injected antigen analyte. In this example, the response at 90 seconds was plotted against the concentration of injected HSA creating a standard curve so that the concentration of HSA in unknown samples could be calculated. Known control samples were also injected.
The experimental conditions are summarized in the following table:
- Ligand: Anti-HSA
- Analyte: HSA
- Analyte Concentrations: 40, 20, 10, 5, 2.5,1.25, 0.626, 0.3125, 0.1562, 0.781, 0.0391, 0.0195nM
- Association Time: 3 min
- Dissociation Time: 3 min
- Regeneration: 10 mM Glycine pH 2 with 10% Glycerol
The SR7500DC SPR system monitors this antibody-antigen interaction in real-time with simultaneous measurements of sample and reference channels.