ABSTRACT
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 microM 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 0.1-1mM, 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.
Subject(s)
Antibodies, Monoclonal/immunology , Carbocyanines/analysis , Fatty Acids/analysis , Fatty Acids/immunology , Fluorescent Dyes/metabolism , Spectrometry, Fluorescence/methods , Surface Plasmon Resonance/methods , Animals , Antibody Specificity , Binding, Competitive , Carbocyanines/chemistry , Fatty Acids/chemistry , Fatty Acids/metabolism , Kinetics , MiceABSTRACT
Competitive time-resolved fluoroimmunoassays (FIAs) were developed for measuring 1,3,5(10)-estratrien-3-ol-17-one glucosiduronate (estrone 3-glucuronide, E(1)3G) and 5 beta-Pregnane-3 alpha,20 alpha-diol 3-glucosiduronate (pregnanediol 3-glucuronide, Pd3G) in unextracted urine. The assays are specific, detect 0.98 ng E(1)3G/mL and 0.035 microgram Pd3G/mL, measure 102.8 +/- 2.0% of E(1)3G and 93.6 +/- 2.9% of Pd3G added, and exhibit between and within assay coefficients of variation, respectively, of 5.3% and 7.1% for E(1)3G and 6.8% and 7.8% for Pd3G. The urine matrix does not interfere with the assay. Urinary steroid glucuronide profiles measured by these FIAs conform to those of urinary steroid glucuronides and serum estradiol and progesterone measured by other established immunoassays. These FIAs afford the advantages of non-radioisotopic procedures and urine sample collection (convenience, non-invasiveness, integration of pulsatile secretion) to evaluate menstrual function in epidemiological, medical, and athletic populations.
Subject(s)
Estrogens, Conjugated (USP)/urine , Estrone/analogs & derivatives , Pregnanediol/analogs & derivatives , Antibodies, Monoclonal , Cross Reactions , Estrone/urine , Female , Fluoroimmunoassay , Humans , Immunoenzyme Techniques , Male , Menstrual Cycle/physiology , Pregnanediol/immunology , Pregnanediol/urine , Radioimmunoassay , Reference Values , Reproducibility of Results , Sensitivity and Specificity , Time FactorsABSTRACT
Unlike antibodies produced in rabbits and other animals, mouse antibodies have proved to be difficult to covalently label with fluorochromes. By careful control of the labelling conditions, a method has been established for labelling mouse Thy-1 antibodies obtained by immunisation with normal thymus cells. The resultant FITC Thy-1 antibodies suffer little loss in cytotoxic properties, react with the correct specificity and can be used to label Thy-1 positive cells.
