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  - 
TY  - JOUR
A1  - Schenk, Jörg A.
A1  - Sellrie, Frank
A1  - Böttger, Volker
A1  - Micheel, Burkhard
A1  - Stöcklein, Walter F. M.
T1  - Generation and application of a fluorescein-specific single chain antibody
N2  - A recombinant single chain antibody fragment (designated scDE1) of the murine monoclonal anti-fluorescein antibody B13-DE1 was generated using the original hybridoma cells as source for the variable antibody heavy and light chain (VH and VL) genes. After cloning the variable genes into a phage vector a functional antibody fragment was selected by phage display panning. Recombinant antibody could be expressed as phage antibody and as soluble single chain antibody in Escherichia coli. High yield of scDE1 could also be detected in bacterial culture supernatant. The scDE1 showed the same binding specificity as the parental monoclonal antibody, i.e. it bound fluorescein, fluorescein derivatives and a fluorescein peptide mimotope. Surface plasmon resonance revealed a K(D) of 19 nM for the scDE1 compared to 0.7 nM for the monoclonal antibody. The isolated soluble scDE1 could easily be conjugated to horseradish peroxidase which allowed the use of the conjugate as universal indicator for the detection of fluorescein-labelled proteins in different immunoassays. Detection of hCG in urine was performed as a model system using scDE1. In addition to E. coli the scFv genes could also be transferred and expressed in eukaryotic cells. Finally, we generated HEK293 cells expressing the scDE1 at the cell surface.
Y1  - 2007
UR  - http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VRJ-4P3DY33- 1&_user=1584062&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000053886&_version=1&_urlVersion=0&_userid=1584062&md5=e 4
ER  -