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Principles of ligand capture

Chemistry

High affinity capture relies on a tight interaction between the ligand and a capturing molecule immobilized on the sensor surface. Conditions for capture will vary according to the characteristics of the ligand and capturing molecule. Regeneration normally removes both ligand and bound analyte, and fresh ligand is captured for each analysis cycle. This simplifies optimization of regeneration conditions, although increasing the consumption of ligand.

It is important to choose a capturing molecule which does not interact with the analyte. For kinetic analyses, it is important that the ligand dissociates as little as possible from the surface during measurement, and also that the ligand capture step is highly reproducible so that conditions for each cycle are comparable.

Examples of capturing molecules

• Streptavidin (available on Sensor Chip SA): Requires modification of the ligand (biotinylation). Very high affinity capture, negligible dissociation. The ligand cannot be removed by regeneration.
• Anti-mouse Ig antibodies: Useful for capturing monoclonal antibodies, regardless of antibody specificity.
• Ligand-specific antibodies: Useful for capturing the ligand of interest.
• Protein A, protein G, protein L etc: Useful for capture of antibodies.
• Tag-specific capture: Antibodies or other high-affinity binders directed towards a protein tag are useful for capturing recombinant proteins.

Immobilizing the capturing molecule

Immobilize the capturing molecule on the sensor surface using an appropriate covalent coupling method. The amount of capturing molecule immobilized can be quite high and is not critical for the application.

Sensor Chip SA is a ready-to-use sensor chip with immobilized streptavidin for capture of biotinylated ligands.

Capturing the ligand

Inject ligand solution over the sensor surface. Vary the concentration of ligand in solution to adjust the amount of ligand captured. In cases where the capturing reaction is sufficiently slow, the amount captured can also be adjusted by varying the contact time.

Capturing approaches may be preferable to covalent immobilization in a number of respects:

• The ligand is usually not modified in any way, provided that the capturing interaction does not introduce conformational changes in the ligand. The approach can be valuable for ligands that are easily inactivated by chemical coupling methods.
• Ligand is attached to the surface in a specific orientation (determined by the location of the binding site for the capturing molecule) so that attachment to the surface does not introduce heterogeneity in the ligand population.
• Capturing provides a micro-scale affinity purification of the ligand, allowing immobilization of specific ligands from complex mixtures such as cell extracts or cell culture media.
• Capturing allows the same surface to be used for analysis of interactions involving different ligands, for example in studies of panels of antibodies, since fresh ligand is captured for each cycle.

Capturing approaches consume more ligand when fresh material has to be captured for each analysis cycle, and the maximum attainable analyte binding capacity is usually lower with capture than with direct immobilization of the ligand. In addition, stringent demands are placed on the tightness of the capturing interaction for applications like kinetic determination where dissociation of captured ligand during the analysis cycle complicates evaluation of the results. The advantages of capturing however outweigh these considerations in a wide variety of application situations.

The commonest capturing approaches are streptavidin- or avidin-biotin capture, antibody-based capture, and capture of tagged proteins. Capturing may however in principle be applied to any ligand for which a high affinity interactant is available that binds independently of the analyte.

See also

List of help files

Biotinylation for streptavidin-biotin capture

Capturing tagged recombinant proteins

Antibody-based capture

Streptavidin-biotin capture