Fast selection of antibodies without antigen purification: adaptation of the protein fragment complementation assay to select antigen-antibody pairs.
We have adapted the protein fragment complementation assay (PCA) to the screening and selection of antibodies in the single-chain Fv (scFv) format. In this assay, two interacting proteins (target and antibody) are genetically fused to the two halves of the dissected enzyme dihydrofolate reductase. Binding of the two partners reassembles this enzyme and reconstitutes its activity, thus allowing growth on minimal medium. We have optimized this system with regard to linker length and orientation, and can reach an efficiency for antigen/antibody interactions similar to that with fused leucine zippers. Using several model antibodies specific for peptides and proteins, we show that cognate interactions give rise to about seven orders of magnitude more colonies than non-specific interactions. When transforming mixtures of plasmids encoding different antigens and/or antibodies, all colonies tested contained plasmids encoding cognate pairs. We believe that this system will be very powerful as a routine system for generating antibodies, especially in functional genomics, since it does not require purification and immobilization of the antigen. The identification of an antibody specific for a cDNA or EST-encoded protein will require only cloning, transformation and plating of bacteria.
Mössner
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E
Biochemisches Institut der Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Koch
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H
Plückthun
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A
D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity; D000941:Antigens / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D003001:Cloning, Molecular / Q000379:methods*; D019281:Dimerization; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D005816:Genetic Complementation Test; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D051379:Mice; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology*; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D014295:Trimethoprim / Q000494:pharmacology
D000906:Antibodies; D000941:Antigens; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase