Identification of Successful Variants Based on Binding Properties

Identification of Successful Variants Based on Binding Properties 

In practice, panning methods can also select for undesirable properties. One problem with the use of immobilized ligands is that the immobilization process may alter their structure and binding properties. Moreover, nonspecific binding can occur, such as adsorption to the support material. Both of these problems lead to selection of undesired properties. 

One approach that can overcome immobilization-related problems involves the use of antigen (or ligand) covalently bound to biotin (Ag-B). The Ag-B is added to the solution at the desired concentration to allow solution-phase binding, so that the selection process is based mainly on the differential affinity of each antibody or other binding molecule. The mixture is then added to strepavidin-coated beads or wells, where the formation of the biotin-strepavidin noncovalent complex allows the selection of high affinity antigen-binding molecules. 

Enzymes have been successfully enriched from libraries by selecting variants that bind to transition state analogs, or by covalent trapping with surface bound suicide inhibitors,20 but this approach does not usually select the most active enzymatic variants. 

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