HOOK EFFECT DETERMINATION METHOD FOR HOMOGENEOUS TIME-RESOLVED FLUORESCENCE IMMUNOASSAY

摘要

The present application relates to a hook effect determination method for a homogeneous time-resolved fluorescence immunoassay. The method comprises the steps of: (1) measuring, under the same excitation wavelength, fluorescence intensities of a fluorescence donor and acceptor at two detection wavelengths, and using a fluorescence intensity ratio between the two wavelengths as a signal value; (2) respectively establishing, by means of measuring signal values of at least two time instances in a reaction initiation phase and performing linear fitting and four-parameter fitting, reference curves of an absolute signal intensity value, a signal change rate value, and a sample concentration; and (3) detecting a signal value of an unknown sample, and obtaining, by means of the reference curves, a concentration of the unknown sample according to a fluorescence intensity of the unknown sample, so as to avoid the hook effect. The invention enables calculation of a relative signal intensity and a relative signal change rate value by means of ratios of the absolute signal intensity value and signal change rate value to background signals, thereby achieving consistency of results across different instruments. The invention increases the number of sample concentration readings which can be completed within a given period of time, thereby improving the detection throughput of systems.