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Download fileEvaluation of Nanobody Conjugates and Protein Fusions as Bioanalytical Reagents
journal contribution
posted on 2017-03-20, 00:00 authored by Virginia
J. Bruce, Brian R. McNaughtonEnzyme-linked
immunosorbent assay (ELISA), flow cytometry, and
Western blot are common bioanalytical techniques. Successful execution
traditionally requires the use of one or more commercially available
antibody–small-molecule dyes or antibody–reporter protein
conjugates that recognize relatively short peptide tags (<15 amino
acids). However, the size of antibodies and their molecular complexity
(by virtue of post-translational disulfide formation and glycosylation)
typically require either expression in mammalian cells or purification
from immunized mammals. The preparation and purification of chemical
dye– or reporter protein–antibody conjugates is often
complicated and expensive and not commonplace in academic laboratories.
In response, researchers have developed comparatively simpler protein
scaffolds for macromolecular recognition, which can be expressed with
relative ease in E. coli and can be evolved to bind
virtually any target. Nanobodies, a minimalist scaffold generated
from camelid-derived heavy-chain IgGs, are one such example. A multitude
of nanobodies have been evolved to recognize a diverse array of targets,
including a short peptide. Here, this peptide tag (termed BC2T) and
BC2 nanobody–dye conjugates or reporter protein fusions are
evaluated in ELISA, flow cytometry, and Western blot experiments and
compared to analogous experiments using commercially available antibody–conjugate/peptide
tag pairs. Collectively, the utility and practicality of nanobody-based
reagents in bioanalytical chemistry is demonstrated.
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Keywords
peptide tagBC 2Tdyenanobody-based reagentsProtein FusionsBioanalytical Reagents Enzyme-linked immunosorbent assayflow cytometryWestern blotbioanalytical chemistryantibodybioanalytical techniquesWestern blot experimentsELISANanobody Conjugatesprotein scaffoldsconjugateSuccessful executioncamelid-derived heavy-chain IgGsreporter protein fusionspost-translational disulfide formationpurification