Abstract: A method for detecting multiple subpopulations of analytes of interest in a sample employing a complementary binding moiety to each of said analytes bound to a solid support, wherein each analyte and its complementary binding moiety comprise first and second members of a specific binding pair (msbp) respectively is provided. The method includes the steps of forming a mixture of known proportions of multiple subpopulations of said complementary binding moieties, wherein each subpopulation comprises a different complementary binding moieties, contacting the sample with the mixture so that specific binding pairs are formed on the solid supports, and relating the presence of analytes of interest in the sample to the formation of specific binding pairs associated with each unique proportion of said multiple subpopulations. The method can be performed with solid supports of a single average size and a single fluorochrome and without the need for using three detection systems (fluorescence FS & SS).