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A robust pipeline for rapid production of versatile nanobody repertoires
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A robust pipeline for rapid production of versatile nanobody repertoires
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Journal Article
A robust pipeline for rapid production of versatile nanobody repertoires
2014
Overview
This paper presents an efficient method for generating nanobodies with high affinity and high specificity. In addition, a collection of nanobodies specific for GFP or mCherry that resulted from this work is described.
Nanobodies are single-domain antibodies derived from the variable regions of Camelidae atypical immunoglobulins. They show promise as high-affinity reagents for research, diagnostics and therapeutics owing to their high specificity, small size (∼15 kDa) and straightforward bacterial expression. However, identification of repertoires with sufficiently high affinity has proven time consuming and difficult, hampering nanobody implementation. Our approach generates large repertoires of readily expressible recombinant nanobodies with high affinities and specificities against a given antigen. We demonstrate the efficacy of this approach through the production of large repertoires of nanobodies against two antigens, GFP and mCherry, with
K
d
values into the subnanomolar range. After mapping diverse epitopes on GFP, we were also able to design ultrahigh-affinity dimeric nanobodies with
K
d
values as low as ∼30 pM. The approach presented here is well suited for the routine production of high-affinity capture reagents for various biomedical applications.
Publisher
Nature Publishing Group US,Nature Publishing Group
Subject
/ 82
/ 82/1
/ 82/58
/ Animals
/ Biomedical Engineering/Biotechnology
/ Green Fluorescent Proteins - immunology
/ Green Fluorescent Proteins - metabolism
/ Luminescent Proteins - immunology
/ Luminescent Proteins - metabolism
/ Proteins
/ Reagents
/ Recombinant Proteins - genetics
/ Recombinant Proteins - isolation & purification
/ Recombinant Proteins - metabolism
/ Single-Domain Antibodies - genetics
/ Single-Domain Antibodies - immunology
/ Single-Domain Antibodies - isolation & purification
/ Single-Domain Antibodies - metabolism
/ Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
/ Studies
