Principles of Affinity Selection

Abstract

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.