TY  - JOUR
A1  - Eisold, Ursula
A1  - Kupstat, Annette
A1  - Klier, Dennis Tobias
A1  - Primus, Philipp-A.
A1  - Pschenitza, Michael
A1  - Niessner, Reinhard
A1  - Knopp, Dietmar
A1  - Kumke, Michael Uwe
T1  - Probing the physicochemical interactions of 3-hydroxy-benzo[a]pyrene with different monoclonal and recombinant antibodies by use of fluorescence line-narrowing spectroscopy
JF  - Analytical & bioanalytical chemistry
N2  - Characterization of interactions between antigens and antibodies is of utmost importance both for fundamental understanding of the binding and for development of advanced clinical diagnostics. Here, fluorescence line-narrowing (FLN) spectroscopy was used to study physicochemical interactions between 3-hydroxybenzo[a]pyrene (3OH-BaP, as antigen) and a variety of solvent matrices (as model systems) or anti-polycyclic aromatic hydrocarbon antibodies (anti-PAH). We focused the studies on the specific physicochemical interactions between 3OH-BaP and different, previously obtained, monoclonal and recombinant anti-PAH antibodies. Control experiments performed with non-binding monoclonal antibodies and bovine serum albumin (BSA) indicated that nonspecific interactions did not affect the FLN spectrum of 3OH-BaP. The spectral positions and relative intensities of the bands in the FLN spectra are highly dependent on the molecular environment of the 3OH-BaP. The FLN bands correlate with different vibrational modes of 3OH-BaP which are affected by interactions with the molecular environment (pi-pi interactions, H-bonding, or van-der-Waals forces). Although the analyte (3OH-BaP) was the same for all the antibodies investigated, different binding interactions could be identified from the FLN spectra on the basis of structural flexibility and conformational multiplicity of the antibodies' paratopes.
KW  - FLNS
KW  - Antibody
KW  - Paratope
KW  - Hapten
KW  - Polycyclic aromatic hydrocarbons
Y1  - 2014
U6  - https://doi.org/10.1007/s00216-013-7584-8
SN  - 1618-2642
SN  - 1618-2650
VL  - 406
IS  - 14
SP  - 3387
EP  - 3394
PB  - Springer
CY  - Heidelberg
ER  - 
TY  - JOUR
A1  - Kupstat, Annette
A1  - Kumke, Michael Uwe
A1  - Hildebrandt, Niko
T1  - Toward sensitive, quantitative point-of-care testing (POCT) of protein markers miniaturization of a homogeneous time-resolved fluoroimmunoassay for prostate-specific antigen detection
JF  - The analyst : the analytical journal of the Royal Society of Chemistry
N2  - Point-of-care testing (POCT) systems which allow for a sensitive, quantitative detection of protein markers are extremely useful for the early detection and therapy progress monitoring of cancer. However, currently commercially available POCT devices are mainly limited to the qualitative detection of protein markers. In this study we demonstrate the successive miniaturization of a sensitive and fast assay for the quantitative detection of prostate-specific antigen (PSA) using a well established and clinically approved homogeneous time-resolved fluoroimmunoassay technology (TRACE (R)) on a commercial plate-reader system (KRYPTOR (R)). Regarding the initial requirements for the development of POCT devices we applied a 30-fold assay volume reduction (150 mu L to 5 mu L) to achieve a reasonable lab-on-a-chip volume and a 24-fold and 120-fold excitation pulse energy reduction to achieve reasonable pulse energies for low-cost miniature excitation sources. Due to highly efficient optimization of key POCT parameters our miniaturized PSA assay achieved a 30% increased sensitivity and a 2-fold improved limit of detection compared to the standard plate-reader method. Our results demonstrate the successful implementation of key parameters for a significant miniaturization and for cost reduction in the clinically approved KRYPTOR (R) platform for protein detection. The technological alterations required are easy-to-implement and can be immediately adapted for more than 30 diagnostic protein markers already available for the KRYPTOR (R) platform. These features strongly recommend our assay format to be utilized in innovative, sensitive, quantitative POCT of protein markers.
Y1  - 2011
U6  - https://doi.org/10.1039/c0an00684j
SN  - 0003-2654
VL  - 136
IS  - 5
SP  - 1029
EP  - 1035
PB  - Royal Society of Chemistry
CY  - Cambridge
ER  - 
TY  - THES
A1  - Kupstat, Annette
T1  - Förster resonance energy transfer (FRET)-based detection of molecular binding interactions - from fundamental research to application in life science
Y1  - 2012
CY  - Potsdam
ER  -