TY  - JOUR
A1  - Eisold, Ursula
A1  - Sellrie, Frank
A1  - Schenk, Jörg A.
A1  - Lenz, Christine
A1  - Stöcklein, Walter F. M.
A1  - Kumke, Michael Uwe
T1  - Bright or dark immune complexes of anti-TAMRA antibodies for adapted fluorescence-based bioanalysis
JF  - Analytical & bioanalytical chemistry
N2  - 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.
KW  - mAb
KW  - Fluorescence
KW  - Anisotropy
KW  - Exciplex
KW  - Energy-transfer probe
Y1  - 2015
U6  - https://doi.org/10.1007/s00216-015-8538-0
SN  - 1618-2642
SN  - 1618-2650
VL  - 407
IS  - 12
SP  - 3313
EP  - 3323
PB  - Springer
CY  - Heidelberg
ER  -