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Analytical and Bioanalytical Chemistry

, Volume 407, Issue 12, pp 3313–3323 | Cite as

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

  • Ursula Eisold
  • Frank Sellrie
  • Jörg A. Schenk
  • Christine Lenz
  • Walter F. M. Stöcklein
  • Michael U. KumkeEmail author
Research Paper

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.

Graphical Abstract

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

Keywords

mAb Fluorescence Anisotropy Exciplex Energy-transfer probe 

Notes

Acknowledgement

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

Supplementary material

216_2015_8538_MOESM1_ESM.pdf (737 kb)
ESM 1 (PDF 737 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Ursula Eisold
    • 1
  • Frank Sellrie
    • 2
  • Jörg A. Schenk
    • 2
    • 3
  • Christine Lenz
    • 2
  • Walter F. M. Stöcklein
    • 4
  • Michael U. Kumke
    • 1
    Email author
  1. 1.Department of Chemistry, Physical ChemistryUniversität PotsdamPotsdamGermany
  2. 2.UP Transfer GmbHPotsdamGermany
  3. 3.Hybrotec GmbHPotsdamGermany
  4. 4.Fraunhofer Institute for Cell Therapy and Immunology (IZI)PotsdamGermany

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