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Bright or dark immune complexes of anti-TAMRA antibodies for adapted fluorescence-based bioanalysis

  • Fluorescence labels, for example fluorescein or rhodamin derivatives, are widely used in bioanalysis applications including lateral-flow assays, PCR, and fluorescence microscopy. Depending on the layout of the particular application, fluorescence quenching or enhancement may be desired as the detection principle. Especially for multiplexed applications or high-brightness requirements, a tunable fluorescence probe can be beneficial. The alterations in the photophysics of rhodamine derivatives upon binding to two different anti-TAMRA antibodies were investigated by absorption and fluorescence-spectroscopy techniques, especially determining the fluorescence decay time and steady-state and time-resolved fluorescence anisotropy. Two monoclonal anti-TAMRA antibodies were generated by the hybridoma technique. Although surface-plasmon-resonance measurements clearly proved the high affinity of both antibodies towards 5-TAMRA, the observed effects on the fluorescence of rhodamine derivatives were very different. Depending on the anti-TAMRAFluorescence labels, for example fluorescein or rhodamin derivatives, are widely used in bioanalysis applications including lateral-flow assays, PCR, and fluorescence microscopy. Depending on the layout of the particular application, fluorescence quenching or enhancement may be desired as the detection principle. Especially for multiplexed applications or high-brightness requirements, a tunable fluorescence probe can be beneficial. The alterations in the photophysics of rhodamine derivatives upon binding to two different anti-TAMRA antibodies were investigated by absorption and fluorescence-spectroscopy techniques, especially determining the fluorescence decay time and steady-state and time-resolved fluorescence anisotropy. Two monoclonal anti-TAMRA antibodies were generated by the hybridoma technique. Although surface-plasmon-resonance measurements clearly proved the high affinity of both antibodies towards 5-TAMRA, the observed effects on the fluorescence of rhodamine derivatives were very different. Depending on the anti-TAMRA antibody either a strong fluorescence quenching (G71-DC7) or a distinct fluorescence enhancement (G71-BE11) upon formation of the immune complex was observed. Additional rhodamine derivatives were used to gain further information on the binding interaction. The data reveal that such haptens as 5-TAMRA could generate different paratopes with equal binding affinities but different binding interactions, which provide the opportunity to adapt bioanalysis methods including immunoassays for optimized detection principles for the same hapten depending on the specific requirements.show moreshow less

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Author details:Ursula EisoldORCiDGND, Frank Sellrie, Jörg A. SchenkORCiD, Christine Lenz, Walter F. M. Stöcklein, Michael Uwe KumkeORCiDGND
DOI:https://doi.org/10.1007/s00216-015-8538-0
ISSN:1618-2642
ISSN:1618-2650
Pubmed ID:https://pubmed.ncbi.nlm.nih.gov/25711988
Title of parent work (English):Analytical & bioanalytical chemistry
Publisher:Springer
Place of publishing:Heidelberg
Publication type:Article
Language:English
Year of first publication:2015
Publication year:2015
Release date:2017/03/27
Tag:Anisotropy; Energy-transfer probe; Exciplex; Fluorescence; mAb
Volume:407
Issue:12
Number of pages:11
First page:3313
Last Page:3323
Organizational units:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Chemie
Peer review:Referiert
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