title: Colony assays for antibody fragments expressed in bacteria.
abstract: This paper describes procedures for the detection and selection of bacterial colonies expressing antibody fragments of desired antigen specificity. Fab and Fv fragments are detected in a filter assay in which bacterial colonies are grown on a master filter in contract with a second, antigen-coated filter. Ab fragments diffusing onto the second filter bind antigen directly and specifically and are detected with a monoclonal antibody directed against a myc-tag sequence fused to the carboxy-terminal end of the light chain or heavy chain (direct assay). Single-chain Fv (scFv) in which the VH and V1 sequences are joined by a short linker peptide are detected by a modified procedure in which scFv are immobilized on filters coated with the anti-myc-tag sequence and subsequently detected by specific binding to radiolabeled antigen (indirect assay). A single positive bacterial colony expressing antigen-specific Fv (or scFv) can be recovered among at least 10,000 negative colonies using the procedures described. The direct assay has been successfully used to discriminate Fv fragments which express point mutations known to increase the binding affinity of antibodies to the hapten 2-phenyl-oxazolone. The procedures described may thus prove generally useful for the selection of antigen-specific clones expressed in bacteria and/or higher-affinity variants of such antibodies.
authors.affiliation: Laboratory of Molecular Biology, Cambridge, U.K.
authors.forename: M L
authors.initials: ML
authors.lastname: Dreher
authors.forename: E
authors.initials: E
authors.lastname: Gherardi
authors.forename: A
authors.initials: A
authors.lastname: Skerra
authors.forename: C
authors.initials: C
authors.lastname: Milstein
mesh_terms: D000906:Antibodies / Q000235:genetics*; D000915:Antibody Affinity; D000918:Antibody Specificity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005803:Genes, Immunoglobulin; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007163:Immunosorbent Techniques; D010081:Oxazolone / Q000276:immunology; D010957:Plasmids; D011994:Recombinant Proteins / Q000276:immunology
chemical_list: D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D010081:Oxazolone