The purpose of the present invention is to provide: a method for detecting a substance of interest and a method for quantifying a substance of interest, whereby it becomes possible to detect or quantify the substance of interest properly using a reagent that can be produced with high yield; a kit; and a method for preparing a regent.A method for detecting a substance of interest in a sample comprises a step of mixing a first conjugate 10, a second conjugate 20 and the sample together, then placing the resultant mixture under conditions such that a stimulus-responsive substance 11 can agglomerate, and then determining the presence or absence of the occurrence of dispersion of the stimulus-responsive substance 11 or the occurrence of an event associating with the aforementioned dispersion, wherein the first conjugate 10 is a conjugate of a first substance that contains the stimulus-responsive substance 11 with a first affinity substance 13 for a substance of interest 50, and the second conjugate 20 is a conjugate of a second substance 21 that has a hydrophilic moiety or an electrically charged moiety with a second affinity substance 23 for the substance of interest 50. The second substance 21 contains particles having a specific gravity of 1.4 or more, and the first affinity substance 13 and the second affinity substance 23 can bond to the substance of interest 50 simultaneously at different sites in the substance of interest 50 from each other.
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