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.
Galanis
Maria
M
Cooperative Research Center for Diagnostic Technologies at CSIRO Molecular Science, Parkville, Victoria, Australia.
Irving
Robert A
RA
Cooperative Research Center for Diagnostic Technologies at CSIRO Molecular Science, Parkville, Victoria, Australia.
Hudson
Peter J
PJ
Cooperative Research Center for Diagnostic Technologies at CSIRO Molecular Science, Parkville, Victoria, Australia.
D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001435:Bacteriophages / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000821:virology*; D005822:Genetic Vectors / Q000235:genetics / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000276:immunology; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D019151:Peptide Library; D011994:Recombinant Proteins; C055966:immunoglobulin Fv