Chemistry: analytical and immunological testing – Biospecific ligand binding assay
Reexamination Certificate
1998-06-04
2001-05-15
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Biospecific ligand binding assay
C436S014000, C436S147000, C436S164000, C436S172000, C436S095000, C436S531000, C436S545000, C436S546000, C436S805000, C436S807000, C436S815000, C436S827000, C435S007800, C435S007100, C435S007930, C435S014000, C424S009362
Reexamination Certificate
active
06232130
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to the use of low valency carbohydrate binding ligands to evaluate a carbohydrate in a sample, e.g., using such ligands with fluorescence resonance energy transfer (FRET) to measure carbohydrate, e.g., free carbohydrate or a carbohydrate of a carbohydrate containing compound.
SUMMARY OF THE INVENTION
In general, the invention features, a method of evaluating a carbohydrate in a sample. The method includes:
providing a low valency carbohydrate binding ligand,
providing a glycoconjugate, which preferably includes a label, and a carbohydrate moiety,
contacting the low valency carbohydrate binding ligand and the glycoconjugate with the sample,
determining the extent of binding of the low valency carbohydrate binding ligand with the glycoconjugate
the binding of the low valency carbohydrate binding ligand with the glycoconjugate being correlated with the amount of carbohydrate in the sample.
The label, when present, can be any substance which allows detection of the glycoconjugate, e.g., a radioactive label, a fluorescent label, an enzyme, a proximity-based signal generating label moiety, e.g., a FRET component, a homogeneous time resolved fluorescence (HTRF) component, a luminescent oxygen channeling assay (LOCI) component, biotin or avidin or other functionally similar substances, an antibody moiety recognized by an antibody or an antibody, or an antigen binding portion of antibody.
In preferred embodiments, the evaluation is made in the presence of the glycoconjugate displaced by the carbohydrate and the low valency carbohydrate binding ligand reversibly bound to the carbohydrate.
In preferred embodiments, the sample includes a body fluid; a urine sample; a blood sample; a plasma sample; intracellular fluid; interstitial fluid; or a cell homogenate or extract.
In preferred embodiments, the method measures the concentration of glucose in a sample which includes subcutaneous body fluids, intracutaneous body fluid, or blood.
In preferred embodiments, the carbohydrate analyte is a monosaccharide; a disaccharide; a polysaccharide; glucose; a carbohydrate which is a component of another molecule or supramolecular structures, e.g., a macromolecule, e.g., a carbohydrate moiety of a glycoprotein.
In preferred embodiments, the glycoconjugate includes one or more glycosylated serum albumin, preferably human or bovine, capable of binding to low valency carbohydrate binding ligands.
In preferred embodiments, the low valency carbohydrate binding ligand is a lectin, e.g., reduced valency Concanavalin A.
In preferred embodiments, the low valency carbohydrate binding ligand and the glycoconjugate bind reversibly.
In preferred embodiments, the low valency carbohydrate binding ligand and the glycoconjugate bind reversibly over the range of carbohydrate concentrations found in body fluids; physiological concentrations of carbohydrate in the body fluid being tested, e.g., 0.05 mg/ml to 5.0 mg/ml or 0.5 mg/ml to 5 mg/ml of carbohydrates, e.g., glucose.
In another aspect, the invention features, a method of evaluating a carbohydrate in a sample. The method includes the steps of:
a) contacting the sample with a specific binding pair which includes a first binding member and a second binding member,
wherein the first binding member includes a low valency carbohydrate binding ligand coupled to a first energy absorbing FRET component,
wherein the second binding member is a glycoconjugate which includes a carbohydrate and a second energy absorbing FRET component,
wherein the excited state energy level of the first energy absorbing FRET component overlaps with the excited state energy level of the second energy absorbing FRET component, and
wherein the low valency carbohydrate binding ligand and the glycoconjugate can reversibly bind to each other such that carbohydrate present in the sample can displace the glycoconjugate and can reversibly bind to the low valency carbohydrate binding ligand; and
b) evaluating the extent to which non-radiative fluorescence resonance energy transfer occurs between the first energy absorbing FRET component and the second energy absorbing FRET component,
thereby evaluating the carbohydrate in the sample.
In preferred embodiments, the evaluation is made in the presence of the glycoconjugate displaced by the carbohydrate and the low valency carbohydrate binding ligand reversibly bound to the carbohydrate.
Energy transfer can, by way of example, be evaluated by measuring one or more of: donor quenching, donor lifetime, e.g., decrease in donor excited lifetime, sensitized acceptor emission, fluorescence depolarisation. It can also be measured by determining the ratio of two parameters, e.g., the ratio of a donor parameter to an acceptor parameter, e.g., the ratio of donor to acceptor fluorescence, or depolarization of fluorescence relative to excitation.
In preferred embodiments, the evaluation includes measuring energy transfer as a function of fluorescence intensities of the first energy absorbing FRET component and the second energy absorbing FRET component.
In preferred embodiments, the method includes comparing the result of step (b) with a FRET value obtained from a calibration step.
In preferred embodiments, the sample includes a body fluid; a urine sample; a blood sample; a plasma sample; intracellular fluid; interstitial fluid; or a cell homogenate or extract.
In preferred embodiments, the method measures the concentration of glucose in a sample which includes subcutaneous body fluid, intracutaneous body fluid, or blood.
In preferred embodiments, the carbohydrate analyte is a monosaccharide; a disaccharide; a polysaccharide; glucose; a carbohydrate which is a component of another molecule or supramolecular structures, e.g., a macromolecule, e.g., a carbohydrate moiety of a glycoprotein.
In preferred embodiments, the glycoconjugate includes one or more glycosylated serum albumin, preferably human or bovine, capable of binding to low valency carbohydrate binding ligands.
In preferred embodiments, the low valency carbohydrate binding ligand is a lectin, e.g., reduced valency Concanavalin A.
In preferred embodiments, the low valency carbohydrate binding ligand and the glycoconjugate bind reversibly over the range of carbohydrate concentrations found in body fluids; physiological concentrations of carbohydrate in the body fluid being tested, e.g., 0.05 mg/ml to 5.0 mg/ml or 0.5 mg/ml to 5 mg/ml of carbohydrates, e.g., glucose.
In preferred embodiments, first and second energy absorbing FRET component are fluorophores and, at least one of fluorophores is chosen from the group consisting of fluoresceins, rhodamines, BODIPY's, cyanine dyes and phycobiliproteins; the low valency carbohydrate binding ligand is labeled with a fluorophore and the glycoconjugate is labeled with a fluorophore in the non-radiative fluorescence resonance energy transfer process; low valency carbohydrate binding ligand is labeled with a fluorophore which is the acceptor and the glycoconjugate is labeled with a fluorophore which is the donor in the non-radiative fluorescence resonance energy transfer process.
In preferred embodiments, the first member of the specific binding pair is fluorophore-labeled low valency Concanavalin A, the second member of the specific binding pair is fluorophore-labeled glycosylated serum albumin capable of binding to low valency Concanavalin A, and the non-radiative fluorescence resonance energy transfer can be determined by measuring the ratio of the light emissions attributable to the two fluorophores.
In preferred embodiments, the specific binding pair is placed in a microdialysis vessel and then subcutaneously implanted in the subject; the specific binding pair is encapsulated and then subcutaneously implanted in the subject; the specific binding pair is mixed with a carrier, e.g., an oil into which glucose can pass and then implanted, e.g., injected subcutaneously, in the subject; the specific binding pair is tattooed into the skin of the subject; the components of the specific binding pair are chemically modified t
Fish & Richardson P.C.
Gabel Gailene R.
Le Long V.
Sensor Technologies Inc.
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