Biacore X100 reference portal

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.