Methods for conducting multiplexed assays

A method of conducting a multiplexed binding assay for a plurality of analytes comprising a first analyte and a second analyte of interest comprising

• (a) combining, in one or more steps, the following components:
• (i) a sample comprising the plurality of analytes comprising the first analyte of interest and the second analyte of interest,
• (ii) a first targeting agent immobilized on a first binding domain, wherein the first targeting agent comprises an oligonucleotide,
• (iii) a first targeting agent complement connected to a first linking agent, wherein the first targeting agent complement comprises an oligonucleotide complementary to the first targeting agent oligonucleotide, forming a first oligonucleotide pair,
• (iv) a first binding reagent connected to a first supplemental linking agent, wherein the first binding reagent is a binding partner of the first analyte,
• (v) a second targeting agent immobilized on a second binding domain, wherein the second targeting agent comprises an oligonucleotide,
• (vi) a second targeting agent complement connected to a second linking agent, wherein the second targeting agent complement comprises an oligonucleotide complementary to the second targeting agent oligonucleotide, forming a second oligonucleotide pair,
• (vii) a second binding reagent connected to a second supplemental linking agent, wherein the second binding reagent is a binding partner of the second analyte, and
• (viii) at least two copies of a bridging agent,
• wherein,
• a first copy of the bridging agent has a first binding site for the first linking agent and an additional binding site for the first supplemental linking agent, and a second copy of the bridging agent has a first binding site for the second linking agent and an additional binding site for the second supplemental linking agent;
• (b) forming
• (i) a first binding complex on the first binding domain comprising the first targeting agent, the first targeting agent complement, the first linking agent, the first copy of the bridging agent, the first supplemental linking agent, the first binding reagent and the first analyte, and
• (ii) a second binding complex on the second binding domain comprising the second targeting agent, the second targeting agent complement, the second linking agent, the second copy of the bridging agent, the second supplemental linking agent, the second binding reagent and the second analyte, and
• (c) measuring the amount of the first and second analytes on the first and second binding domains, respectively.