TY - JOUR
T1 - Surface plasmon resonance-enhanced fluorescence implementation of a single-step competition assay: Demonstration of fatty acid measurement using an anti-fatty acid monoclonal antibody and a Cy5-labeled fatty acid
AU - Vareiro, Mmlm
AU - Tranchant, I
AU - Maplin, S
AU - Zak, K
AU - Gani, M M
AU - Slevin, C J
AU - Hailes, H C
AU - Tabor, A B
AU - Cameron, P J
AU - Jenkins, A T A
AU - Williams, D E
N1 - ID number: ISI:000255918000019
PY - 2008
Y1 - 2008
N2 - The development of a single-step, separation-free method for measurement of low concentrations of fatty acid using a surface plasmon resonance-enhanced fluorescence competition assay with a surface-bound antibody is described. The assay behavior was unexpectedly complex. A nonlinear coverage-dependent self-quenching of emission from surface-bound fluorescent label was deduced from the response kinetics and attributed to a surface plasmon-mediated energy transfer between adsorbed fluorophores, modified by the effects of plasmon interference. Principles of assay design to avoid complications from such effects are discussed. An anti-fatty acid mouse monoclonal antibody reacting to the alkyl chain was prepared and supported on a gold chip at a spacing appropriate for surface-plasmon field-enhanced fluorescence spectroscopy (SPEFS), by applying successively a self-assembled biotinylated monolayer, then streptavidin, then biotinylated protein A, and then the antibody, which was crosslinked to the protein A. Synthesis of a fluorescently (Cy5) tagged C-11 fatty acid is reported. SPEFS was used to follow the kinetics of the binding of the labeled fatty acid to the antibody, and to implement a competition assay with free fatty acid (undecanoic acid), sensitive at the 1 mu M scale, a sensitivity limit caused by the low affinity of antibodies for free fatty acids, rather than the SPEFS technique itself. Free fatty acid concentration in human serum is in the range 0.1-1 mM, suggesting that this measurement approach could be applied in a clinical diagnostic context. Finally, a predictive, theoretical model of fatty acid binding was developed that accounted for the observed "overshoot" kinetics. (c) 2008 Elsevier Inc. All rights reserved.
AB - The development of a single-step, separation-free method for measurement of low concentrations of fatty acid using a surface plasmon resonance-enhanced fluorescence competition assay with a surface-bound antibody is described. The assay behavior was unexpectedly complex. A nonlinear coverage-dependent self-quenching of emission from surface-bound fluorescent label was deduced from the response kinetics and attributed to a surface plasmon-mediated energy transfer between adsorbed fluorophores, modified by the effects of plasmon interference. Principles of assay design to avoid complications from such effects are discussed. An anti-fatty acid mouse monoclonal antibody reacting to the alkyl chain was prepared and supported on a gold chip at a spacing appropriate for surface-plasmon field-enhanced fluorescence spectroscopy (SPEFS), by applying successively a self-assembled biotinylated monolayer, then streptavidin, then biotinylated protein A, and then the antibody, which was crosslinked to the protein A. Synthesis of a fluorescently (Cy5) tagged C-11 fatty acid is reported. SPEFS was used to follow the kinetics of the binding of the labeled fatty acid to the antibody, and to implement a competition assay with free fatty acid (undecanoic acid), sensitive at the 1 mu M scale, a sensitivity limit caused by the low affinity of antibodies for free fatty acids, rather than the SPEFS technique itself. Free fatty acid concentration in human serum is in the range 0.1-1 mM, suggesting that this measurement approach could be applied in a clinical diagnostic context. Finally, a predictive, theoretical model of fatty acid binding was developed that accounted for the observed "overshoot" kinetics. (c) 2008 Elsevier Inc. All rights reserved.
UR - http://www.scopus.com/inward/record.url?scp=43049097468&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2008.03.033
DO - 10.1016/j.ab.2008.03.033
M3 - Article
SN - 0003-2697
VL - 377
SP - 243
EP - 250
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -