Assays for Selection of Single-Chain Fragment Variable Recombinant Antibodies to Metal Nanoclusters
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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.
Affiliation(s): (2) Department of Biochemistry, Vanderbilt University, Nashville, TN
(3) Department of Chemistry, Vanderbilt University, Nashville, TN
(3) Department of Chemistry, Vanderbilt University, Nashville, TN
Book Title: NanoBiotechnology Protocols
Series: Methods in Molecular Biology | Volume: 303 | Pub. Date: Apr-27-2005 | Page Range: 113-120 | DOI: 10.1385/1-59259-901-X:113
Subject: Biotechnology
Key Words: Single-chain fragment variable (ScFv) - recombinant antibodies - horseradish peroxidase - Anti-E tag antibody - silver-coated membrane - nanoparticles
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