Method and composition for the simultaneous and discrete analysis of multiple analytes

A method for simultaneously detecting multiple analytes of interest in a sample, said method comprising:

• (a) combining said sample with a composition comprising a population of particulate supports that are detectable by flow cytometry techniques, wherein
• A. each particulate support consists essentially of an unlabeled particle to which is bound exactly one of a set of at least two unlabeled specific reagents, each of said specific reagents is capable of binding specifically to one of the multiple analytes of interest,
• B. the population comprises discrete subpopulations of supports, each of which consists of those supports comprising the same specific reagent, wherein the combined number of supports in any two or more subpopulations is unique as compared to the number of supports in any other combination of subpopulations or as to a single subpopulation,
• C. each subpopulation constitutes a predetermined, known proportion of the population of particulate supports, and
• D. the particles of the population are substantially physically indistinguishable from each other, the particulate supports being of approximately the same mean diameter, whereby in the presence of one or more analytes a discrete population of specific binding pars is formed on the supports of each subpopulation for each analyte of interest in the sample;
• (b) contacting said specific binding parts with a labeled agent, said labeled agent comprising a number of specific binding moieties each attached to a fluorochrome which emits a detectable fluorescence upon exposure to excitation energy, wherein each of the binding moieties is specific for one of the multiple analytes and the same fluorochrome is attached to each of the binding moieties;
• (c) removing any unbound labeled agent and detecting fluorescence intensity of each particulate support of a preselected number of said population of particulate supports using flow cytometry techniques;
• (d) obtaining a histogram plot of said preselected number of particulate supports detected as a function of the logarithm of said fluorescence intensity detected, said histogram plot contains one or more peaks, wherein each peak has an area which indicates the proportion of each subpopulation of particulate supports of said preselected number associated with said peak, and wherein each peak has a position for each analyte detected which may be the same as or different from the peak position of any other analyte detected and wherein absence of one or more analytes results in a peak having a position at essentially background fluoroescence;
• and
• (e) identifying analytes present in said simple by the relative proportion of each peak as a function of the relative proportion of said preselected supports.