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

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Abstract

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-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.

The fluorescence of 5-TAMRA is altered upon antibody binding. Depending on the antibody used the fluorescence is heavily quenched (left) or enhanced (right) by the binding interaction

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Acknowledgement

The authors wish to thank Dietmar Knopp for providing the C6 ET probe.

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Authors and Affiliations

  1. Department of Chemistry, Physical Chemistry, Universität Potsdam, Karl-Liebknecht-Str. 24-25, 14476, Potsdam, Germany

    Ursula Eisold & Michael U. Kumke

  2. UP Transfer GmbH, Am Neuen Palais 10, 14469, Potsdam, Germany

    Frank Sellrie, Jörg A. Schenk & Christine Lenz

  3. Hybrotec GmbH, Am Mühlenberg 11, 14476, Potsdam, Germany

    Jörg A. Schenk

  4. Fraunhofer Institute for Cell Therapy and Immunology (IZI), Am Mühlenberg 13, 14476, Potsdam, Germany

    Walter F. M. Stöcklein

Authors
  1. Ursula Eisold
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  2. Frank Sellrie
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  3. Jörg A. Schenk
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  4. Christine Lenz
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  5. Walter F. M. Stöcklein
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  6. Michael U. Kumke
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Correspondence to Michael U. Kumke.

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Eisold, U., Sellrie, F., Schenk, J.A. et al. Bright or dark immune complexes of anti-TAMRA antibodies for adapted fluorescence-based bioanalysis. Anal Bioanal Chem 407, 3313–3323 (2015). https://doi.org/10.1007/s00216-015-8538-0

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  • DOI: https://doi.org/10.1007/s00216-015-8538-0

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