| Popis: |
A method to screen and isolate antigen specific clones from a library of single-chain antibodies expressed on the surface of yeast cells is presented. Two rounds of magnetic bead enrichment before flow cytometric sorting enables one to screen libraries of far greater diversity than can be screened by just flow cytometry. The strength of flow cytometric sorting is the ability to follow the selection in real time and to isolate easily the highest affinity antigen-specific clones. A major strength of yeast display as a discovery platform is the ability to characterize the binding properties, the affinity of a clone without the need for subcloning, expression, and purification of the scFv. The methodology for directed evolution of single-chain antibodies to increase the affinity of a clone is also described. |
