Maria Galanis

Bacteriophage Library Construction and Selection of Recombinant Antibodies

This unit describes the use of E. coli and bacteriophages to display a diverse library of antibody fragments equivalent in complexity to the mammalian immune repertoire, and subsequent screening of the library for antibody fragments with specific binding affinities. The methods are also used for affinity enhancement (maturation), through the display and selection of improved affinity mutants derived from a single parent antibody. This unit discusses the following key components needed in library construction technology: a repertoire of antibody genes, typically amplified by polymerase chain reaction (PCR) technology; construction of scFv genes by PCR assembly; a method for producing a stable library, using bacteriophage that can both display individual antibodies on the viral surface and carry the gene encoding the antibody; a method of growing phage for selection; a method of selecting the highest‐affinity antibody from the phage library; a method for monitoring progress of phage selection; an affinity‐enhancement strategy for improving and manipulating the selected antibody; and expression of affinity‐enhanced antibodies.