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Site-Specific Photoconjugation of Beta-Lactamase Fragments to Monoclonal Antibodies Enables Sensitive Analyte Detection via Split-Enzyme Complementation
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.ORCID iD: 0000-0003-4214-6991
2018 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 13, no 7, article id 1700688Article in journal (Refereed) Published
Abstract [en]

Protein fragment complementation assays (PCA) rely on a proximity-driven reconstitution of a split reporter protein activity, typically via interaction between bait and prey units separately fused to the reporter protein halves. The PCA principle can also be formatted for use in immunossays for analyte detection, e.g., via the use of small immunoglobulin binding proteins (IgBp) as fusion partners to split-reporter protein fragments for conversion of pairs of antibodies into split-protein half-probes. However, the non-covalent binding between IgBp and antibodies is not ideal for development of robust assays. Here, the authors describe how split-enzyme reporter halves can be both site-specifically and covalently photoconjugated at antibody Fc-parts for use in homogeneous dual-antibody in vitro immunoassays based on analyte-dependent split-enzyme fragment complementation. The half-probes consist of parts of a beta-lactamase split-protein reporter fused to an immunoglobulin Fc binding domain equipped with a unique cysteine residue at which a photoactivable maleimide benzophenone group (MBP) is attached. Using such antibody conjugates the authors obtain an analyte-driven complementation of the reporter enzyme fragments monitored via conversion of a chromogenic substrate. Results from detection of human interferon-gamma and the extracellular domain of HER2 is shown. The described principles for site-specific conjugation of proteins to antibodies should be broadly applicable.

Place, publisher, year, edition, pages
WILEY-V C H VERLAG GMBH , 2018. Vol. 13, no 7, article id 1700688
Keywords [en]
affibody, antibody, complementation, homogeneous assay, nitrocefin, photoconjugation, split-beta-lactamase
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-232401DOI: 10.1002/biot.201700688ISI: 000437281600007PubMedID: 29485240Scopus ID: 2-s2.0-85043698701OAI: oai:DiVA.org:kth-232401DiVA, id: diva2:1235647
Funder
VINNOVA
Note

QC 20180726

Available from: 2018-07-26 Created: 2018-07-26 Last updated: 2018-07-26Bibliographically approved

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Yu, FeifanAlesand, VeronicaNygren, Per-Åke

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