Method of agglutination immunoassay
The present invention provides a particle enhanced agglutination immunoassay including the steps of: mixing a sample solution containing an analyte with a solution containing insoluble carrier particles carrying a binding partner or binding partners for the analyte to prepare a mixed solution; determining a variation (i) in intensity of light scattered from the mixed solution based on a difference in intensity of scattered light between first and second time points; determining a variation (ii) in absorbance of the mixed solution based on a difference in absorbance between third and fourth time points; and correlating the determined variation (i) in intensity of scattered light and the determined variation (ii) in absorbance with an amount of the analyte present in the sample using a calibration curve plotted based on the variation in intensity of scattered light and a calibration curve plotted based on the variation in absorbance. The present invention employs measurements of the intensity of scattered light and the absorbance in combination for a single assay, and thus provides a particle enhanced agglutination immunoassay which achieves higher sensitivity and a wider dynamic range than conventional assays.
1. A particle enhanced agglutination immunoassay comprising the steps of:
mixing a sample solution containing an analyte with a solution containing insoluble carrier particles carrying a binding partner or binding partners for the analyte to prepare a mixed solution;
determining a variation (i) in intensity of light scattered from the mixed solution based on a difference in intensity of scattered light between a first and a second time point;
determining a variation (ii) in absorbance of the mixed solution based on a difference in absorbance between a third and a fourth time point; and
correlating the determined variation (i) in intensity of scattered light and the determined variation (ii) in absorbance with an amount of the analyte present in the sample using a calibration curve plotted based on the variation in intensity of scattered light and a calibration curve plotted based on the variation in absorbance,
wherein at a low concentration of analyte, the concentration is determined based on the calibration curve plotted based on the measurement of the intensity of scattered light and at a high concentration of analyte, the concentration is determined based on the calibration curve plotted based on the measurement of the absorbance; and
wherein the time period which is defined by the third and fourth time points and for which the variation (ii) is determined is shorter than the time period which is defined by the first and second time points and for which the variation (i) is determined, and the determining a variation (i) in intensity light scattered and the determining a variation (ii) in absorbance are combined in a single assay.
2. The immunoassay according to claim 1 , wherein the first, second, third, and fourth time points are respectively selected between 0 and 1000 seconds after the start of preparation of the mixed solution.
3. The immunoassay according to claim 1 , wherein the variations (i) and (ii) are determined at the same wavelength.
4. The immunoassay according to claim 1 , wherein the variation (ii) in absorbance is determined at a wavelength within a range of ±25% of a wavelength at which the variation (i) in intensity of scattered light is determined.
5. The immunoassay according to claim 4 , wherein the variation (ii) in absorbance is determined at two wavelengths consisting of main and sub-wavelengths which are shorter and longer, respectively, than the wavelength at which the variation (i) in intensity of scattered light is determined.
6. The immunoassay according to claim 1 , wherein the variations (i) and (ii) are determined at a wavelength within the range of from 550 to 900 nm.
7. The immunoassay according to claim 1 , wherein said calibration curves cover at least a range consisting of said low concentration of analyte and said high concentration of analyte.