A Tri-part Protein Complementation System Using Antibody-Small Peptide Fusions Enables Homogeneous Immunoassays

Abstract Protein-fragment complementation is a valuable tool for monitoring protein interactions. In complementation assays, the reporter fragments are directly fused to the interacting proteins, eliminating the possibility of monitoring native interactions. In principle, complementation could be ac...

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Autores principales: Andrew S. Dixon, Sun Jin Kim, Brett K. Baumgartner, Sylvia Krippner, Shawn C. Owen
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Publicado: Nature Portfolio 2017
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Acceso en línea:https://doaj.org/article/97ac8ae7398d48fb8a4f94d4d926ddba
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spelling oai:doaj.org-article:97ac8ae7398d48fb8a4f94d4d926ddba2021-12-02T11:40:51ZA Tri-part Protein Complementation System Using Antibody-Small Peptide Fusions Enables Homogeneous Immunoassays10.1038/s41598-017-07569-y2045-2322https://doaj.org/article/97ac8ae7398d48fb8a4f94d4d926ddba2017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-07569-yhttps://doaj.org/toc/2045-2322Abstract Protein-fragment complementation is a valuable tool for monitoring protein interactions. In complementation assays, the reporter fragments are directly fused to the interacting proteins, eliminating the possibility of monitoring native interactions. In principle, complementation could be achieved by placing the reporter fragments on antibodies which bind to the proteins of interest, enabling the monitoring of endogenous protein interactions or detection of a single protein in a homogeneous immunoassay. Previous reports have demonstrated proof-of-concept of this approach; however, current complementation systems have not met the practical requirements as suitable fusion partners for antibodies while providing the sensitivity needed for immunoassays. To surmount these challenges, we created a first-in-class, tri-part split luciferase consisting of two 11-residue peptides that are used as the antibody appendages. As an initial proof-of-concept, we used antibody-peptide fusions and found them to be capable of quantifying pg/mL concentrations of soluble or cell-bound HER2, proving this unique complementation system overcomes previous limitations and transforms this approach from merely possible to practical and useful. As shown herein, this dual-peptide system provides a rapid, simple, and sensitive “add-and-read” homogeneous immunoassay platform that can be broadly adapted as an alternative to traditional immunoassays, and in the future should enable complementation to be expanded to monitoring endogenous protein interactions.Andrew S. DixonSun Jin KimBrett K. BaumgartnerSylvia KrippnerShawn C. OwenNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-13 (2017)
institution DOAJ
collection DOAJ
language EN
topic Medicine
R
Science
Q
spellingShingle Medicine
R
Science
Q
Andrew S. Dixon
Sun Jin Kim
Brett K. Baumgartner
Sylvia Krippner
Shawn C. Owen
A Tri-part Protein Complementation System Using Antibody-Small Peptide Fusions Enables Homogeneous Immunoassays
description Abstract Protein-fragment complementation is a valuable tool for monitoring protein interactions. In complementation assays, the reporter fragments are directly fused to the interacting proteins, eliminating the possibility of monitoring native interactions. In principle, complementation could be achieved by placing the reporter fragments on antibodies which bind to the proteins of interest, enabling the monitoring of endogenous protein interactions or detection of a single protein in a homogeneous immunoassay. Previous reports have demonstrated proof-of-concept of this approach; however, current complementation systems have not met the practical requirements as suitable fusion partners for antibodies while providing the sensitivity needed for immunoassays. To surmount these challenges, we created a first-in-class, tri-part split luciferase consisting of two 11-residue peptides that are used as the antibody appendages. As an initial proof-of-concept, we used antibody-peptide fusions and found them to be capable of quantifying pg/mL concentrations of soluble or cell-bound HER2, proving this unique complementation system overcomes previous limitations and transforms this approach from merely possible to practical and useful. As shown herein, this dual-peptide system provides a rapid, simple, and sensitive “add-and-read” homogeneous immunoassay platform that can be broadly adapted as an alternative to traditional immunoassays, and in the future should enable complementation to be expanded to monitoring endogenous protein interactions.
format article
author Andrew S. Dixon
Sun Jin Kim
Brett K. Baumgartner
Sylvia Krippner
Shawn C. Owen
author_facet Andrew S. Dixon
Sun Jin Kim
Brett K. Baumgartner
Sylvia Krippner
Shawn C. Owen
author_sort Andrew S. Dixon
title A Tri-part Protein Complementation System Using Antibody-Small Peptide Fusions Enables Homogeneous Immunoassays
title_short A Tri-part Protein Complementation System Using Antibody-Small Peptide Fusions Enables Homogeneous Immunoassays
title_full A Tri-part Protein Complementation System Using Antibody-Small Peptide Fusions Enables Homogeneous Immunoassays
title_fullStr A Tri-part Protein Complementation System Using Antibody-Small Peptide Fusions Enables Homogeneous Immunoassays
title_full_unstemmed A Tri-part Protein Complementation System Using Antibody-Small Peptide Fusions Enables Homogeneous Immunoassays
title_sort tri-part protein complementation system using antibody-small peptide fusions enables homogeneous immunoassays
publisher Nature Portfolio
publishDate 2017
url https://doaj.org/article/97ac8ae7398d48fb8a4f94d4d926ddba
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