Back to page index


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.



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.

Panel 2 header

Lorem ipsum dolor sit amet, consectetur adipisicing elit. Repellendus aliquid tempore natus voluptates repudiandae, eos dolorem libero inventore, quod incidunt, asperiores. Reiciendis itaque enim pariatur, blanditiis minima autem quo a nulla, obcaecati quis, excepturi atque ab rerum! Sequi molestias vel eum, hic, perspiciatis eius suscipit reprehenderit molestiae vitae similique.

  ${panel2ButtonCTA}

Panel 3 header

Lorem ipsum dolor sit amet, consectetur adipisicing elit. Repellendus aliquid tempore natus voluptates repudiandae, eos dolorem libero inventore, quod incidunt, asperiores. Reiciendis itaque enim pariatur, blanditiis minima autem quo a nulla, obcaecati quis, excepturi atque ab rerum! Sequi molestias vel eum, hic, perspiciatis eius suscipit reprehenderit molestiae vitae similique.

  ${panel3ButtonCTA}

Panel 4 header

Lorem ipsum dolor sit amet, consectetur adipisicing elit. Repellendus aliquid tempore natus voluptates repudiandae, eos dolorem libero inventore, quod incidunt, asperiores. Reiciendis itaque enim pariatur, blanditiis minima autem quo a nulla, obcaecati quis, excepturi atque ab rerum! Sequi molestias vel eum, hic, perspiciatis eius suscipit reprehenderit molestiae vitae similique.

Panel 4 header 2

Lorem ipsum dolor sit amet, consectetur adipisicing elit. Repellendus aliquid tempore natus voluptates repudiandae, eos dolorem libero inventore, quod incidunt, asperiores. Reiciendis itaque enim pariatur, blanditiis minima autem quo a nulla, obcaecati quis, excepturi atque ab rerum! Sequi molestias vel eum, hic, perspiciatis eius suscipit reprehenderit molestiae vitae similique.

Panel 5 header

Lorem ipsum dolor sit amet, consectetur adipisicing elit. Repellendus aliquid tempore natus voluptates repudiandae, eos dolorem libero inventore, quod incidunt, asperiores. Reiciendis itaque enim pariatur, blanditiis minima autem quo a nulla, obcaecati quis, excepturi atque ab rerum! Sequi molestias vel eum, hic, perspiciatis eius suscipit reprehenderit molestiae vitae similique.

Panel 5 header 2

Lorem ipsum dolor sit amet, consectetur adipisicing elit. Repellendus aliquid tempore natus voluptates repudiandae, eos dolorem libero inventore, quod incidunt, asperiores. Reiciendis itaque enim pariatur, blanditiis minima autem quo a nulla, obcaecati quis, excepturi atque ab rerum! Sequi molestias vel eum, hic, perspiciatis eius suscipit reprehenderit molestiae vitae similique.

Panel 5 header 3

Lorem ipsum dolor sit amet, consectetur adipisicing elit. Repellendus aliquid tempore natus voluptates repudiandae, eos dolorem libero inventore, quod incidunt, asperiores. Reiciendis itaque enim pariatur, blanditiis minima autem quo a nulla, obcaecati quis, excepturi atque ab rerum! Sequi molestias vel eum, hic, perspiciatis eius suscipit reprehenderit molestiae vitae similique.