Other

University System of Maryland

Becton Dickinson and Company Limited

Badri P Maliwal

Richard B Thompson

Vincent L Feliccia

Daniel Elbaum

Joseph R Lakowicz

Marcia W Patchan

David W Christianson

Zhengfang Ge

Alvydas J Ozinskas

Carol A Fierke

Peter A Koen

The invention relates to compositions and kits for homogeneous fluorescence polarization (anisotropy) assays for detecting and quantifying metal ions in solution. Metal-dependent binding of a fluorescent ligand to an unlabeled macromolecule effects a measurable change in anisotropy as will the binding of metal ions to a fluorescent labeled macromolecule. Binding of the fluorescent ligand to the unlabeled macromolecule is metal dependent with the change in anisotropy being proportional to the concentration of bound metal ions. Conversely, if the fluorescent label is first conjugated to a macromolecule and the macromolecule is subsequently stripped of metal ion, it may then be used to signal binding of metal ions. The covalently bound fluorescent label exhibits changes in anisotropy proportional to the concentration of bound metal ions. Kits comprise a fluorescent molecule and a macromolecule.

Determination of metal ions in solution by photoluminescence anisotropy

Homogeneous fluorescence polarization (anisotropy) assays for detecting and quantifying metal ions in solution, based the metal-dependent binding of a fluorescent ligand to an unlabeled macromolecule, or the binding of a metal ion to a fluorescent labeled macromolecule. The metal-dependent binding of a fluorescent ligand to an unlabeled macromolecule (metallo-macromolecule) effects a measurable change in anisotropy as will the binding of metal ions to a fluorescent labeled macromolecule. Binding of the fluorescent ligand to the unlabeled macromolecule is metal dependent with the change in anisotropy being proportional to the concentration of bound metal ions. No binding of the fluorescent ligand to the macromolecule occurs in the absence of metal ions. Conversely, if the fluorescent label is first conjugated to a metallo-macromolecule and the metallo-macromolecule is subsequently stripped of its metal ion, it may then be used to transduce the binding of metal ions. Transduction is provided wherein the covalently bound fluorescent label exhibits changes in anisotropy proportional to the concentration of bound metal ions. In all methods, the change in anisotropy may be simply related to the metal ion concentration of the test solution.

The invention described in detail herein relates to the detection, determination, and quantitation of certain ions and small molecules in solution. The invention specifically relates to improvements in the area of photoluminescent sensors for use in a detection scheme involving the alteration of a photoluminescent label or moiety attached to or associated with an analyte binding macromolecule. One may use the changes in photoluminescence lifetime, changes in ratios of photoluminescence intensity or changes in photoluminescence polarization (anisotropy) to determine the analyte. The photoluminescence change measured correlates to the concentration of the ion or molecule in solution.

Photoluminescent sensors of chemical analytes

A fluorometric luminescence immunoassay method includes forming a sample by exposing a first immune reaction reactant to a second immune reaction reactant capable of reacting with the first reactant, one of the first and second immune reaction reactants being labelled with a photoluminescent energy transfer donor and the other being labelled with a photoluminescent energy transfer acceptor complementary to the photoluminescent donor. At least the photoluminescent donor has the property of photoluminescence, and the photoluminescent donor and acceptor are chosen so that when the first immune reaction reactant reacts with the second immune reaction reactant, the donor and the acceptor are capable of interacting to produce a detectable luminescence lifetime change. The sample is excited with radiation, and the resulting emission is detected. The apparent luminescent lifetime is then calculated to determine the presence of a reaction product of the first and second immune reaction reactants.

Fluorescent energy transfer immunoassay

A method for measuring the concentration of a saccharide, conjugated saccharide or polysaccharide of interest using luminescent lifetimes and energy transfer in which an energy transfer donor-acceptor pair is added to a sample to be analyzed, the donor of the donor-acceptor pair being photoluminescent. The acceptor is bound to a carrier, while the donor and any saccharide, conjugated saccharide or polysaccharide of interest present in the sample compete for binding sites on the carrier. The sample is irradiated and the resultant emission detected. Energy transfer occurs between the donors and the acceptors, which produces a detectable lifetime change of the fIuorescence of the donor. The lifetime change is reduced or even eliminated by the competitive binding of a saccharide, conjugated saccharide or polysaccharide of interest to the donor. By measuring the apparent luminescent lifetime, the amount of a saccharide, conjugated saccharide or polysaccharide of interest in the sample can be determined.

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Baltimore, MD, United States of America

Badri P Maliwal