A Tri-part Protein Complementation System Using Antibody-Small Peptide Fusions Enables Homogeneous Immunoassays
Autor: | Sun Jin Kim, Sylvia Krippner, Andrew S. Dixon, Brett K. Baumgartner, Shawn C. Owen |
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Jazyk: | angličtina |
Rok vydání: | 2017 |
Předmět: |
0301 basic medicine
Models Molecular Receptor ErbB-2 Recombinant Fusion Proteins Science Protein combining Enzyme-Linked Immunosorbent Assay Plasma protein binding Computational biology Biology Antibodies Article Protein–protein interaction 03 medical and health sciences Structure-Activity Relationship 0302 clinical medicine Protein-fragment complementation assay Cell Line Tumor Protein Interaction Mapping medicine Humans Luciferase Protein Interaction Domains and Motifs Binding site Immunoassay Multidisciplinary Binding Sites medicine.diagnostic_test Genetic Complementation Test Proteins Reproducibility of Results Molecular biology Peptide Fragments Complementation 030104 developmental biology 030220 oncology & carcinogenesis Medicine Protein Binding |
Zdroj: | Scientific Reports, Vol 7, Iss 1, Pp 1-13 (2017) Scientific Reports |
ISSN: | 2045-2322 |
Popis: | 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. |
Databáze: | OpenAIRE |
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