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
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.
antibody variable domains, bacterial receptor domain, taq dna-polymerase, binding-protein, combinatorial libraries, conformational-changes, immunoassay, affibody, display, allows
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:kth:diva-5645DOI: 10.1016/j.ab.2004.07.003ISI: 000224710600007ScopusID: 2-s2.0-4644337415OAI: oai:DiVA.org:kth-5645DiVA: diva2:10082
QC 20100916 QC 201109152006-05-052006-05-052011-09-15Bibliographically approved