Principles of Affinity Selection

The most common application of phage-display technology is the discovery of peptides or proteins that specifically bind some molecule or other substance of interest-for example, antibodies that specifically bind an antigen. The discovery process starts with a library encompassing a very large array of proteins or peptides with a great diversity of binding specificities-for example, single-chain antibodies with a great diversity of antigen-binding sites. Each member of the array is displayed on the surface of hundreds to billions of identical virus particles (virions) belonging to a single-phage clone; the library as a whole comprises millions to billions of such clones, all mixed together in a single vessel. Affinity selection is the process by which a molecule or substance of interest-generically called the selector-is used to select very rare clones in the library displaying proteins or peptides that happen to bind the selector with high affinity and selectivity. Here, I explain general principles guiding a successful affinity-selection project-principles grounded in phage biology, kinetics of reversible binding, technological advances, and the practical experience of thousands of investigators around the globe.