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Fluorescence resonance energy transfer-based detection of analytes using antiidiotypic affinity protein pairs
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0003-4214-6991
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0002-0695-5188
2004 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 334, no 1, 72-80 p.Article in journal (Refereed) Published
Abstract [en]

A new method for specific detection of proteins based on fluorescence resonance energy transfer (FRET) using affinity proteins (affibodies) derived from combinatorial engineering of Staphylococcal protein A has been developed. Antiidiotypic affibody pairs were used in a homogeneous competitive binding assay, where the idiotypic, target-specific affibody was labeled with fluorescein and the antiidiotypic affibody was labeled with tetramethylrhodamine. Intermolecular FRET between the two fluorescent probes was observed in the antiidiotypic affibody complex, but upon addition of target protein the antiidiotypic affibody was displaced, which was monitored by a shift in the relative emission of the donor and acceptor fluorophores. The feasibility of the system was demonstrated by the detection of IgA and Taq DNA polymerase with high specificity, using two different antiidiotypic affibody pairs. Detection of Taq DNA polymerase in 25% human plasma was successfully carried out, demonstrating that the method can be used for analysis of proteins in samples of complex composition.

Place, publisher, year, edition, pages
2004. Vol. 334, no 1, 72-80 p.
Keyword [en]
antibody variable domains, bacterial receptor domain, taq dna-polymerase, binding-protein, combinatorial libraries, conformational-changes, immunoassay, affibody, display, allows
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-5645DOI: 10.1016/j.ab.2004.07.003ISI: 000224710600007Scopus ID: 2-s2.0-4644337415OAI: oai:DiVA.org:kth-5645DiVA: diva2:10082
Note
QC 20100916 QC 20110915Available from: 2006-05-05 Created: 2006-05-05 Last updated: 2011-09-15Bibliographically approved
In thesis
1. Fluorescence-based ligand assays for protein detection using affibody affinity proteins
Open this publication in new window or tab >>Fluorescence-based ligand assays for protein detection using affibody affinity proteins
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The detection and quantification of biomolecules, and proteins in particular, are of great interest since these molecules are of fundamental importance to our well-being. Body fluids, as for instance human blood, are well suited for sampling of protein levels. However, the complexity of the fluids and the low abundance of many of the interesting biomolecules makes detection and quantification difficult. This has spurred an interest into the development of many protein detection methods, and of these, ligand assays have proven particularly suitable. In this thesis, different types of ligand assays for protein detection have been developed using affibody molecules as ligands.

In a first study, a homogeneous competitive detection assay was investigated, based on antiidiotypic affibody molecule pairs and fluorescence resonance energy transfer (FRET) as reporting system. The individual members of two anti-idiotypic affibody pairs, each consisting of a target binding (idiotypic) and an anti-idiotypic affibody ligand, were labeled with a donor fluorophore and an acceptor fluorophore, respectively. Incubation with the two target proteins IgA and Taq DNA polymerase resulted in a concentration dependent decrease in the FRET signal, allowing for target protein detection and quantification. For Taq DNA polymerase, detection in 25% human plasma was also possible in the same concentration span as in buffer.

In a second study, a homogeneous, non-competitive detection system was described. Affibody molecules of 58 amino acids directed against IgA and IgG were produced with chemical synthesis, and two fluorophores capable of FRET were site-specifically introduced. Binding of target protein induced a concentration-dependent change in the relative emission of the two fluorophores, which formed the basis for the detection system.

In two studies, affibody molecules were evaluated and shown to function well as capture ligands on microarrays. Synthetic affibody molecules directed against Taq DNA polymerase and IgA were modified by the introduction of immobilization tags. Specific immobilization via a C-terminal cysteine or a biotin moiety, or random immobilization via amino groups, were studied in protein microarray experiments and SPR-based biosensor studies. The experiments showed that all immobilization chemistries resulted in functional capture molecules. A short spacer was also introduced, situated between the affibody and the cysteine and biotin moieties, which was shown to improve binding for all constructs. Multidomain affibody constructs of up to four N- to C-terminally linked domains were shown to increase the amount of bound target, compared to monomeric affibody ligands. Six dimeric affibody constructs directed against IgA, IgG, IgE, Taq DNA polymerase, TNF-α and insulin, respectively, showed low limits of detections for their targets and little or no cross-reactivity with the other target proteins. Dimeric affibody molecules directed against IgA and TNF-α were also shown to function in a sandwich format with antibodies for detection of targets in buffer and in human serum and plasma. Successful discrimination between normal and IgA-deficient sera showed that affibody molecules could be used for specific detection of protein in highly complex backgrounds on microarrays.

Place, publisher, year, edition, pages
Stockholm: KTH, 2006. 94 p.
Keyword
Affibody molecules, biosensors, FRET, immobilisation, solid-phase synthesis, protein microarrays
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-3936 (URN)91-7178-344-X (ISBN)
Public defence
2006-05-19, FR4, Albanova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00
Opponent
Supervisors
Note
QC 20100916Available from: 2006-05-05 Created: 2006-05-05 Last updated: 2011-12-08Bibliographically approved

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Publisher's full textScopushttp://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4D48XPV-1&_user=4478132&_handle=V-WA-A-W-WD-MsSAYZA-UUA-U-AAVEBWYEUE-AAVZEUEDUE-YVCAAEAUC-WD-U&_fmt=summary&_coverDate=11%2F01%2F2004&_rdoc=7&_orig=browse&_srch=%23toc%236692%232004%23996659998%23522369!&_cdi=6692&view=c&_acct=C000034958&_version=1&_urlVersion=0&_userid=4478132&md5=a46e6e44818e59ef2a6e6be1f1e3597b

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Nygren, Per-ÅkeEriksson Karlström, Amelie

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