Making antibody fragments using phage display libraries.
To by-pass hybridoma technology and animal immunization, we are trying to build antibodies in bacteria by mimicking features of immune selection. Recently we used fd phage to display antibody fragments fused to a minor coat protein, allowing enrichment of phage with antigen. Using a random combinatorial library of the rearranged heavy (VH) and kappa (V kappa) light chains from mice immune to the hapten 2-phenyloxazol-5-one (phOx), we have now displayed diverse libraries of antibody fragments on the surface of fd phage. After a single pass over a hapten affinity column, fd phage with a range of phOx binding activities were detected, at least one with high affinity (dissociation constant, Kd = 10(-8) M). A second pass enriched for the strong binders at the expense of the weak. The binders were encoded by V genes similar to those found in anti-phOx hybridomas but in promiscuous combinations (where the same V gene is found with several different partners). By combining a promiscuous VH or V kappa gene with diverse repertoires of partners to create hierarchical libraries, we elicited many more pairings with strong binding activities. Phage display offers new ways of making antibodies from V-gene libraries, altering V-domain pairings and selecting for antibodies with good affinities.
Clackson
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T
MRC Laboratory of Molecular Biology, Cambridge, UK.
Hoogenboom
H R
HR
Griffiths
A D
AD
Winter
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G
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000235:genetics*; D000915:Antibody Affinity; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods; D015723:Gene Library; D005803:Genes, Immunoglobulin; D006241:Haptens; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D010081:Oxazolone / Q000031:analogs & derivatives / Q000276:immunology; D016133:Polymerase Chain Reaction
D000906:Antibodies; D006241:Haptens; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; C018361:2-phenyloxazolone; D010081:Oxazolone