Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites.
We describe a rapid, eukaryotic, in vitro method for selection and evolution of antibody combining sites using antibody-ribosome-mRNA (ARM) complexes as selection particles. ARMs carrying single-chain (VH/K) binding fragments specific for progesterone were selected using antigen-coupled magnetic beads; selection simultaneously captured the genetic information as mRNA, making it possible to generate and amplify cDNA by single-step RT-PCR on the ribosome-bound mRNA for further manipulation. Using mutant libraries, antigen-binding ARMs were enriched by a factor of 10(4)-10(5)-fold in a single cycle, with further enrichment in repeated cycles. While demonstrated here for antibodies, the method has the potential to be applied equally for selection of receptors or peptides from libraries.
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Laboratory of Molecular Recognition and Translocus Therapeutics Ltd, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK. mingyue.he@bbsrc.ac.uk
Taussig
M J
MJ
D000818:Animals; D000906:Antibodies / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis; D019143:Evolution, Molecular; D015723:Gene Library; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D007145:Immunoglobulin kappa-Chains / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D018189:Immunomagnetic Separation; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011374:Progesterone / Q000276:immunology; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D012270:Ribosomes; D012641:Selection, Genetic
D000906:Antibodies; D018076:DNA, Complementary; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D046911:Macromolecular Substances; D012333:RNA, Messenger; D011374:Progesterone