Direct detection of single molecules on microparticles

A method of analyzing two or more analytes of interest in a biological sample in a single assay, which method comprises:

• (a) capturing a first analyte of interest on a surface of a first microparticle, wherein the first microparticle (i) comprises a plurality of specific binding members immobilized on the surface thereof which bind to the first analyte and (ii) is labeled with a first fluorophore;
• (b) capturing a second analyte of interest on a surface of a second microparticle, wherein (i) the first analyte is different from the second analyte, and (ii) the second microparticle comprises a plurality of specific binding members immobilized on the surface thereof which bind to the second analyte;
• (c) reacting the captured first analyte of interest with a first conjugate, wherein the first conjugate comprises a specific binding member that is labeled with a second fluorophore and binds to the first analyte;
• (d) reacting the captured second analyte with a second conjugate, wherein the second conjugate comprises a specific binding member that is labeled with a third fluorophore and binds to the second analyte, and wherein the first, second, and third fluorophores are different;
• (e) obtaining a transmitted light image of the first and second microparticles;
• (f) obtaining separate fluorescent images of the first and second microparticles corresponding to the first, second, and third fluorophores, respectively;
• and
• (g) analyzing the images.