Patent ID: 7632653

Claim:
A method for detecting the presence or quantity of an analyte residing in a test sample, said method comprising: i) providing a flow-through assay device that comprises a porous membrane in fluid communication with a conjugate pad, the conjugate pad including probes comprising particles modified with a specific binding member configured to bind with the analyte and containing a fluorescent label, said fluorescent label having a fluorescence emission lifetime of greater than about 1 microsecond, said porous membrane defining a detection zone within which is immobilized a capture reagent configured to bind with the analyte, and wherein the porous membrane defines a calibration zone positioned downstream from the detection zone within which is immobilized a capture reagent configured to bind with the probes; ii) contacting the conjugate pad with the test sample and allowing the particles to flow to said detection zone and said calibration zone; iii) subjecting the detection zone to pulses of illumination to generate a detection signal and, after a certain period of time has elapsed following a pulse, measuring the intensity of the detection signal, wherein a fluorescence reader is employed to provide the illumination and measure the intensity of the detection signal, the reader comprising a pulsed excitation source and a time-gated detector; iv) subjecting the calibration zone to pulses of illumination to generate a calibration signal and after a certain period of time has elapsed following a pulse, measuring the intensity of the calibration signal; and v) comparing the intensity of the detection signal to the intensity of the calibration signal, wherein the amount of the analyte within the test sample is proportional to the intensity of the detection signal as calibrated by the calibration signal.