An in vitro method for detecting molecules, employing: a. 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; b. at least two secondary antibodies, wherein at least one of the secondary antibodies is an antibody fragment of a whole immunoglobulin or antigen-binding fragment of a whole immunoglobulin, and 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; c. 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 that are 2 to 9nm or less than 100 kDa from the enzyme; wherein the method comprises carrying out the following steps in order: d. contacting a sample with the at least two primary antibodies; e. contacting the sample with the at least two secondary antibodies; f. performing a wash step; g. contacting the sample with the conjugate; and h. detecting any FRET signal generated by the FRET acceptor, wherein a positive FRET signal will be detected when the FRET donor is on the bound first secondary antibody and the FRET acceptor is on the bound activated conjugate and the FRET donor is located within less than 10 nm from the FRET acceptor, and wherein no FRET signal will be detected when the FRET donor is on the bound first secondary antibody and the FRET acceptor is on the bound activated conjugate and the FRET donor is located greater than 10 nm from the FRET acceptor, or either or both of the first and second sites are not present in the sample. a. 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; b. at least two secondary antibodies, wherein at least one of the secondary antibodies is an antibody fragment of a whole immunoglobulin or antigen-binding fragment of a whole immunoglobulin, and 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; c. 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 that are 2 to 9nm or less than 100 kDa from the enzyme; d. contacting a sample with the at least two primary antibodies; e. contacting the sample with the at least two secondary antibodies; f. performing a wash step; g. contacting the sample with the conjugate; and h. detecting any FRET signal generated by the FRET acceptor,