A chemiluminescent immunoassay for the identification of functional single-chain antibodies

Random mutagenesis of a template single-chain Fv (scFv) antibody generates a mutant library with members of varying ligand affinities; however, only a small population of these mutants exhibit strong (K_d ∼ 1 nM) or improved affinities. We have therefore designed a chemiluminescent sandwich assay which can identify strong ligand-binding 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 colony lifts of these scFvs were screened for hapten-binding activities with the chemiluminescent assay, and only strong binders were detected.