Methods for detecting molecules in a sample

Abstract

The present invention relates to a method for detecting molecules. The method employs: at least two primary antibodies, wherein the first primary antibody binds to a first site on a molecule and the second primary antibody binds to a second site on a molecule, wherein the second site is different from the first site and wherein the first and second primary antibodies are immunologically distinct; at least two secondary antibodies, wherein the first secondary antibody is labelled with a fluorescence resonance energy transfer (FRET) donor and binds to the first primary antibody; and the second secondary antibody is conjugated or fused to an enzyme and binds the second primary antibody, wherein the first secondary antibody does not bind the second primary antibody and the second secondary antibody does not bind the first primary antibody; a conjugate comprising a FRET acceptor and a substrate specific for the enzyme, wherein when the substrate reacts with the enzyme, an activated conjugate forms, which activated conjugate binds to electron rich moieties on a molecular surface adjacent to the enzyme; wherein the method comprises: contacting a sample with the at least two primary antibodies; contacting the sample with the at least two secondary antibodies; performing a wash step; contacting the sample with the conjugate; and detecting any FRET signal generated by the FRET acceptor.