A CHEMILUMINESCENT IMMUNOASSAY FOR THE IDENTIFICATION OF FUNCTIONAL SINGLE-CHAIN ANTIBODIES

Random mutagenesis of a template single-chain Fv (scFv) antibody gener ates a mutant Library with mem members of varying ligand affinities; h owever, only a small population of these mutants exhibit strong (K-d s imilar to 1 nM) or improved affinities. We have therefore designed a c hemiluminescent sandwich assay which can identify strong ligand-bindin g mutants expressed by Escherichia coli. A model system involving scFv 43C9 and selected mutants with varying hapten affinities was used to demonstrate the application of this methodology. Both the plaque and c olony lifts of these scFvs were screened for hapten-binding activities with the chemiluminescent assay, and only strong binders were detecte d. (C) 1995 Academic Press, Inc.