Abstract: This invention provides methods of improving assays for the quantitation of a protein in a sample by enhancing equimolar detection of the protein. Detection is enhanced by modifying the reaction environment of the protein. This invention further provides improved reagent particles for use in the detection and/or quantitation of an analyte in a sample, where the reagent particles comprise two or more coatings of the same or a different substance, such as an antibody, immobilized on an insoluble particle. This invention further provides a method of increasing the sensitivity of particle-enhanced immunoassays by increasing the size of the aggregates that are formed in the assay. Aggregate size is increased by combining the sample with three different antibodies: an analyte-specific antibody immobilized on an insoluble particle, a second antibody specific analyte, and a linker antibody which specifically binds the second antibody but does not bind to the analyte.