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General recommendations for regeneration
Regeneration is the process of removing bound analyte from the sensor chip surface after analysis of a sample, in preparation for the next analysis cycle.
The number of times a sensor surface can be regenerated depends on the nature of the attached ligand, but is frequently greater than 100 and may even be 1000 or more. When the ligand is attached directly to the surface, regeneration removes analyte from the ligand without destroying the ligand activity. When a capturing approach is used, regeneration generally removes both ligand and analyte from the capturing molecule. In this case the stability of the ligand under regeneration conditions is irrelevant. Efficient regeneration is important for successful assays. Incomplete regeneration or loss of the binding activity from the surface will impair the performance of the assay and the useful lifetime of the sensor chip will be shortened. Time spent on establishing suitable regeneration conditions is therefore a valuable investment.
Regeneration of the surface with a regeneration solution is not always necessary: if the analyte dissociates fast enough, all analyte may be removed within a reasonable time simply by washing with buffer. This is immediately evident from the sensorgram, since the response returns to baseline after the sample injection.
The number of times a sensor surface can be regenerated depends on the nature of the attached ligand, but is frequently greater than 100 and may even be 1000 or more. When the ligand is attached directly to the surface, regeneration removes analyte from the ligand without destroying the ligand activity. When a capturing approach is used, regeneration generally removes both ligand and analyte from the capturing molecule. In this case the stability of the ligand under regeneration conditions is irrelevant. Efficient regeneration is important for successful assays. Incomplete regeneration or loss of the binding activity from the surface will impair the performance of the assay and the useful lifetime of the sensor chip will be shortened. Time spent on establishing suitable regeneration conditions is therefore a valuable investment.
Regeneration of the surface with a regeneration solution is not always necessary: if the analyte dissociates fast enough, all analyte may be removed within a reasonable time simply by washing with buffer. This is immediately evident from the sensorgram, since the response returns to baseline after the sample injection.
Even if the nature of the ligand-analyte interaction is probably the major factor determining suitable regeneration conditions, the optimum conditions are affected by the surface properties, ligand density and analyte binding levels.
SENSOR CHIP TYPE
Optimal regeneration conditions can differ slightly for the same ligand-analyte pair on different sensor chip types. In a test study using anti-myoglobin antibodies as ligand and myoglobin as analyte, optimal regeneration with glycine-HCl on Sensor Chip CM3 required pH values 0.2–0.4 units lower than on Sensor Chip CM5.
COUPLING CHEMISTRY
Regeneration properties of the same ligand immobilized on the same sensor chip type can differ slightly with different coupling chemistries.
IMMOBILIZATION LEVEL
The amount of immobilized ligand can have a significant effect on the optimal regeneration conditions. Monoclonal antibodies regenerated with glycine-HCl, for example, have been found to require lower pH values at lower ligand densities. The effect can be as much as 0.5 pH units between 600 and 10,000 RU immobilized antibody.
ANALYTE BINDING LEVEL
The amount of analyte bound to the surface can affect the conditions required for optimal regeneration. In general, higher analyte levels require slightly harsher conditions.
TEMPERATURE
Temperature can have a significant effect on regeneration performance, and it is important that regeneration is optimized and tested at the temperature at which the assay will be run.
SENSOR CHIP TYPE
Optimal regeneration conditions can differ slightly for the same ligand-analyte pair on different sensor chip types. In a test study using anti-myoglobin antibodies as ligand and myoglobin as analyte, optimal regeneration with glycine-HCl on Sensor Chip CM3 required pH values 0.2–0.4 units lower than on Sensor Chip CM5.
COUPLING CHEMISTRY
Regeneration properties of the same ligand immobilized on the same sensor chip type can differ slightly with different coupling chemistries.
IMMOBILIZATION LEVEL
The amount of immobilized ligand can have a significant effect on the optimal regeneration conditions. Monoclonal antibodies regenerated with glycine-HCl, for example, have been found to require lower pH values at lower ligand densities. The effect can be as much as 0.5 pH units between 600 and 10,000 RU immobilized antibody.
ANALYTE BINDING LEVEL
The amount of analyte bound to the surface can affect the conditions required for optimal regeneration. In general, higher analyte levels require slightly harsher conditions.
TEMPERATURE
Temperature can have a significant effect on regeneration performance, and it is important that regeneration is optimized and tested at the temperature at which the assay will be run.
See also
- List of help files
- Choice of regeneration solution
- Determine suitable regeneration conditions
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A AccuracyAffinity - Basic theory Affinity in solution Aldehyde coupling Amine coupling Analyte concentration for kinetic and affinity measurements Assay design for antibody screening Assay design for small molecule screening Assay formats in Biacore systemsAssay setup for concentration analysisAssay setup for regeneration scouting Assay formats for kinetics and affinity Assay setup for kinetics and affinity analysis Assessing solvent correction curves
C Calibration curves for concentration analysis Calibration-free concentration analysis (CFCA) Capture, antibody-based Capturing biotinylated ligands on Sensor Chip CAP Capturing tagged recombinant proteins Causes of poor curve fit Choice of regeneration solution Choice of sensor surface Choosing which interactant to immobilize Curve fitting principles
L Ligand concentration Ligand immobilization levels Ligand thiol coupling Limit of detection (LOD) Limit of quantitation (LOQ) Linearity
M Maleimide coupling chemistry Maleimide coupling using EMCH or BMPH Maleimide coupling using sulfo-GMBS Mass transport and interaction rates Measuring concentration with Biacore
P Performance criteria for concentration measurements Precision Preparation of liposomes for Biacore assays Preparing the active surface for concentration measurement Preparing the active surface for kinetic and affinity measurements Preparing the active surface for specificity measurement Preparing the reference surface for kinetic and affinity measurements Principles of ligand capture Principles of surface regeneration Procedure for immobilization pH scouting Procedure for modification of the ligand with PDEA Procedure for oxidizing the ligand
R Range Reference subtraction Reference surface Regeneration Regeneration scouting procedure Report point position for concentration measurements Robustness
S Sample preparation for kinetic and affinity measurements Sandwich assay for concentration measurements Sensitivity Setting up CFCA measurements Setting up single-cycle kinetics with the Custom Assay Wizard in Biacore X100 Solvent correction Special considerations for concentration measurements Specificity, selectivity and cross-reactivity Standard error or T-value Steady state affinity determination Streptavidin-biotin capture Surface thiol coupling Surface thiol coupling chemistry