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Fluorescence-based ligand assays for protein detection using affibody affinity proteins
KTH, School of Biotechnology (BIO).
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 [en]
Affibody molecules, biosensors, FRET, immobilisation, solid-phase synthesis, protein microarrays
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-3936ISBN: 91-7178-344-X (print)OAI: oai:DiVA.org:kth-3936DiVA: diva2:10086
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
List of papers
1. Fluorescence resonance energy transfer-based detection of analytes using antiidiotypic affinity protein pairs
Open this publication in new window or tab >>Fluorescence resonance energy transfer-based detection of analytes using antiidiotypic affinity protein pairs
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.

Keyword
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:nbn:se:kth:diva-5645 (URN)10.1016/j.ab.2004.07.003 (DOI)000224710600007 ()2-s2.0-4644337415 (Scopus ID)
Note
QC 20100916 QC 20110915Available from: 2006-05-05 Created: 2006-05-05 Last updated: 2017-12-14Bibliographically approved
2. Chemical Synthesis of Triple-Labelled Three-Helix Bundle Binding Proteins for Specific Fluorescent Detection of Unlabelled Protein
Open this publication in new window or tab >>Chemical Synthesis of Triple-Labelled Three-Helix Bundle Binding Proteins for Specific Fluorescent Detection of Unlabelled Protein
Show others...
2005 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, no 6, 1043-1050 p.Article in journal (Refereed) Published
Abstract [en]

Site-specifically triple-labelled three-helix bundle affinity proteins (affibody molecules) have been produced by total chemical Synthesis. The 58 aa affinity proteins were assembled on an automated peptide synthesizer, followed by manual on-resin incorporation of three different reporter groups. An orthogonal protection strategy was developed for the site-specific introduction of 5-(2-aminethylamino)-1-nophthalenesulfonic acid (EDANS) and 6(7-nitrobenzofurazon-4-yiamino)-hexanoic acid (NBDX), constituting a donor/acceptor pair for fluorescence resonance energy transfer (FRET), and a biotin moiety, used for surface immobilization. Circular dichroism and biosensor studies of the synthetic proteins and their recombinant counterparts revealed that the synthetic proteins were folded and retained their binding specificities. The biotin-conjugated protein could be immobilized onto a streptavidin surface without loss of activity. The synthetic, doubly fluorescent-labelled affinity proteins were shown to function as fluorescent biosensors in an assay for the specific detection of unlabelled human IgG and IgA.

Keyword
affinity proteins; biosensors; FRET; peptides; protein modifications; solid-phase synthesis
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-5646 (URN)10.1002/cbic.200400388 (DOI)000229730000015 ()2-s2.0-20444507608 (Scopus ID)
Note
QC 20100715Available from: 2008-12-03 Created: 2008-12-03 Last updated: 2017-12-14Bibliographically approved
3. Affibody protein capture microarrays: synthesis and evaluation of random and directed immobilization of affibody molecules
Open this publication in new window or tab >>Affibody protein capture microarrays: synthesis and evaluation of random and directed immobilization of affibody molecules
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2005 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 341, no 2, 334-343 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules, 58-amino acid three-helix bundle proteins directed to different targets by combinatorial engineering of staphylococcal protein A, were used as capture ligands on protein microarrays. An evaluation of slide types and immobilization strategies was performed to find suitable conditions for microarray production. Two affibody molecules, ZTaq and ZIgA, binding Taq DNA polymerase and human IgA, respectively, were synthesized by solid phase peptide synthesis using an orthogonal protection scheme, allowing incorporation of selective immobilization handles. The resulting affibody variants were used for random surface immobilization (through amino groups) or oriented surface immobilization (through cysteine or biotin coupled to the side chain of Lys58). Evaluation of the immobilization techniques was carried out using both a real-time surface plasmon resonance biosensor system and a microarray system using fluorescent detection of Cy3-labeled target protein. The results from the biosensor analyses showed that directed immobilization strategies significantly improved the specific binding activity of affibody molecules. However, in the microarray system, random immobilization onto carboxymethyl dextran slides and oriented immobilization onto thiol dextran slides resulted in equally good signal intensities, whereas biotin-mediated immobilization onto streptavidin-coated slides produced slides with lower signal intensities and higher background staining. For the best slides, the limit of detection was 3 pM for IgA and 30 pM for Taq DNA polymerase.

Keyword
Affibody; Protein microarray; Immobilization; Biosensor
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-5647 (URN)10.1016/j.ab.2005.03.039 (DOI)000229460800017 ()2-s2.0-19444365959 (Scopus ID)
Note
QC 20100715Available from: 2008-12-03 Created: 2008-12-03 Last updated: 2017-12-14Bibliographically approved
4. Affibody molecules in protein capture microarrays: Evaluation of multidomain ligands and different detection formats
Open this publication in new window or tab >>Affibody molecules in protein capture microarrays: Evaluation of multidomain ligands and different detection formats
Show others...
2007 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 6, no 1, 171-179 p.Article in journal (Refereed) Published
Abstract [en]

The importance of the ligand presentation format for the production of protein capture microarrays was evaluated using different Affibody molecules, produced either as single 6 kDa monomers or genetically linked head-to-tail multimers containing up to four domains. The performances in terms of selectivity and sensitivity of the monomeric and the multidomain Affibody molecules were compared by immobilization of the ligands on microarray slides, followed by incubation with fluorescent-labeled target protein. An increase in signal intensities for the multimers was demonstrated, with the most pronounced difference observed between monomers and dimers. A protein microarray containing six different dimeric Affibody ligands with specificity for IgA, IgE, IgG, TNF-alpha, insulin, or Taq DNA polymerase was characterized for direct detection of fluorescent-labeled analytes. No cross-reactivity was observed and the limits of detection were 600 fM for IgA, 20 pM for IgE, 70 fM for IgG, 20 pM for TNF-alpha, 60 pM for insulin, and 10 pM for Taq DNA polymerase. Also, different sandwich formats for detection of unlabeled protein were evaluated and used for selective detection of IgA or TNF-alpha in human serum or plasma samples, respectively. Finally, the presence of IgA was determined using detection of directly Cy5-labeled normal or IgA-deficient serum samples.

Keyword
affibody molecule, protein microarray, protein capture, multidomain proteins, sandwich assay, taq dna-polymerase, bacterial receptor domain, in-vitro selection, combinatorial libraries, binding-proteins, analyte, cancer, immobilization, immunoassay, antibodies
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-16307 (URN)10.1021/pr060316r (DOI)000243262600017 ()2-s2.0-33846605022 (Scopus ID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved

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