Particulate solid phase immobilized protein quantitation
AssigneeLuminex Corporation
InventorTravis Parker Davies
Life of this patent
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Abstract
A process, a test kit, calibration standards, and the method of preparing such standards as useful for the qualitative and/or quantitative determination of total solid phase- or microparticle-immobilized, amine-containing reactants such as proteins is provided. The operating principle of the invention is distinct from a classical immunoassay based on antibody-antigen immune interaction or from standard calorimetric proteins assays of soluble proteins and allows the detection of as low as attogram (10−18 gm) levels of total analyte per microparticle.
Claims18
A process for determining the relative average amount of at least one protein of interest, which is immobilized on each of a plurality of particulate solid phases, comprising:
- (a) separately subjecting, under substantially the same labeling conditions, a plurality of particulate solid phases, to each of which is covalently immobilized an unknown average amount of at least one protein of interest, and a plurality of standard particulate solid phases to each of which is covalently immobilized a known average amount of at least one reference protein, to labeling conditions effective to provide protein of interest and reference protein both covalently labeled with at least one light-emitting label;
- (b) separating unbound label from the covalently labeled protein of interest and covalently labeled reference protein;
- and
- (c) comparing the average amount of light emitted by labels found on the standard particulate solid phases harboring the reference protein with that emitted by labels found on particulate solid phases harboring the protein of interest to provide a relative average amount of the protein of interest covalently immobilized on each of the particulate solid phases.
A method of quantifying the amount of at least one protein of interest immobilized directly or indirectly on a particulate solid phase comprising:
- (a) separately subjecting one or more particulate solid phases, to each of which is covalently immobilized directly or indirectly an unknown amount of at least one protein of interest, to labeling conditions effective to covalently affix to each particulate solid phase an amount of a light-emitting label which is proportional to the amount of the at least one protein of interest covalently immobilized to the particulate solid phase;
- (b) separating unbound label from the one or more covalently labeled particulate phases;
- and
- (c) relating the amount of light emitted from each particulate solid phase to the amount of the at least one protein of interest covalently immobilized to the particulate solid phase using a standard curve.
A process for determining the presence or amount of an analyte of interest comprising:
- (a) providing a microparticle to which said analyte is covalently bound;
- (b) covalently labeling the microparticle-bound analyte with a light emitting label;
- (c) separating unbound label from the covalently labeled microparticle-bound analyte;
- and
- (d) quantitating labeled analyte by comparing separately to a microparticle preparation standard carrying a known amount of a reference substance covalently labeled with said light-emitting label.
A method of measuring the quantity of an analyte bound to a particulate solid phase, said method comprising the steps of:
- (a) covalently associating the analyte with the particulate solid phase, said particulate solid phase having a fluorescent label embedded within or immobilized thereon, said label being capable of changing a fluorescence signal as a function of the concentration of the analyte;
- (b) providing at least one standard microparticle preparation with the known amount of a reference protein covalently bound thereto, said standard microparticle preparation having the same fluorescent label as in step (a);
- and
- (c) separately measuring the quantity of the analyte covalently associated with the particulate solid phase by comparing the fluorescence signal of said particulate solid phase with the fluorescence signal of the standard microparticle preparation.
A process for determining the relative average amount of at least one protein of interest, which is immobilized on each of a plurality of particulate solid phases, comprising:
- (a) subjecting, under substantially the same labeling conditions, a plurality of particulate solid phases, populations of which are distinguishable from one another to each of which is covalently immobilized an unknown average amount of at least one protein of interest, and a plurality of standard particulate solid phases, populations of which are distinguishable from one another to each of which is covalently immobilized a known average amount of at least one reference protein, to labeling conditions effective to provide protein of interest and reference protein both covalently labeled with at least one light-emitting label;
- and
- (b) comparing by multiplexed flow cytometry the average amount of light emitted by labels found on the standard particulate solid phases harboring the reference protein with that emitted by labels found on particulate solid phases harboring the protein of interest to provide a relative average amount of the protein of interest covalently immobilized on each of the particulate solid phases.
Dependencies are inferred from the claim text.