Assays for Selection of Single-Chain Fragment Variable Recombinant Antibodies to Metal Nanoclusters

Abstract

The protocols herein describe colony-lift and fluorescent immunoassays that were used to identify bacterial colonies that produced single-chain fragment variable (ScFv) recombinant antibodies reactive with zero-state silver. A large (approx 2.9-billion member) phage-displayed antibody library was panned against zero valent silver. Bacterial colonies obtained after two rounds of selection were either lifted onto nitrocellulose filters or picked to individual wells of 384-well microtiter culture plates. Colonies lifted onto filters were placed onto zero valent silvercoated filters and induced to produce soluble ScFv antibodies. ScFv antibodies, expressed by individual colonies, that bound to silver nanocluster-coated filters were detected using an anti-ScFv antibody conjugated to horseradish peroxidase and a chemiluminescent substrate. Colonies picked to 384-well micotiter culture plates were induced to express soluble ScFv antibodies. ScFv antibodies in bacterial periplasmic extract were transferred from 384-well culture plates to 384-well assay plates containing zero-state silver particles and an anti-ScFv antibody conjugated to a fluorescent dye. ScFv antibodies, expressed by individual bacterial clones, that bound to zero valent silver nanoparticles in 384-well assay plates were detected using an FMAT 8100 fluorescent plate reader. The colony-lift and fluorescent immunoassays detected ScFv antibodies reactive with zero valent silver. Similar assay formats should also be useful to detect bacterially expressed recombinant antibodies or proteins to other nanoclusters.