Concentration analysis
Would you like to know the active concentration of your biomolecule?
Biacore technology can be used to accurately determine active concentrations on the basis of a specific interaction with a high affinity binding partner.
A reliable determination of the biologically relevant, active concentration of a biomolecule is important in several fields of quality control in pharmaceutical developement. Heterogeneity of samples can be due to incorrect protein folding, aggregation, chemical or posttranslational modifications of amino acids or the presence of different steroisomers. The use of SPR biosensor technology enables a determination of active concentrations of a biomolecule on the basis of a high affinity interaction with a specific binding partner, allowing rate-based as well as end point measurements.
Calibration curve based assays
- direct binding assay Injection of an analyte on a high density surface. The absolute response is taken after injection and plotted against the concentration. The linear calibration curve is used for calculation of the unkown concentration in the sample. This assay type is suitable for macromolecules (> 5000 Da).
- indirect or competition assay In the solution competition approach a known amount of the detecting molecule is mixed with sample. The concentration of remaining free ligand is determined by relating the binding signal observed to an appropriate standard curve. This method is most useful for low molecular weight molecules.
- In the surface competiton approach the analyte and sample are competing for the same binding site on the surface. The response obtained is inversely related to the analyte concentration in the sample.
Calibration Free Concentration Analysis (CFCA)
- The concentration determination relies on changes in the binding rate of a sample with varying flow rates, when the transport of molecules to the sensor surface is limited by diffusion from the sample. The protein concentration is calculated from the knowledge of the diffusion coefficient of the analyte. This method is useful when no satisfactory calibration standard is available and enables an accurate determination of the active protein concentration, relating to the specific binding activity of the protein.
Please contact our application specialists for getting detailed information and an individual quote.
Tel.: +49 (0) 561-804 4661 | Fax: +49 (0) 561-804 4665 | info@biaffin.de
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