Methods for conducting multiplexed assays

A method of conducting a multiplexed binding assay for a plurality of analytes of interest comprising

• (a) combining, in one or more steps, the following components:
• (i) a sample comprising a first analyte of interest and a second analyte of interest,
• (ii) a first targeting agent immobilized on a first binding domain,
• (iii) a first targeting agent complement connected to a linking agent, wherein the first targeting agent complement is a binding partner of the first targeting agent,
• (iv) a first binding reagent connected to a 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,
• (vi) a second targeting agent complement connected to a linking agent, wherein the second targeting agent complement is a binding partner of the second targeting agent,
• (vii) a second binding reagent connected to a supplemental linking agent, wherein the second binding reagent is a binding partner of the second analyte, and
• (viii) optionally, at least two copies of a bridging agent,
• wherein,
• if the bridging agent is omitted, each linking agent is a binding partner of the supplemental linking agent, or
• if the bridging agent is included, the bridging agent has a first binding site for one of the linking agents and an additional binding site for one of the supplemental linking agents;
• (b) forming
• (i) a first binding complex on the first binding domain comprising the first targeting agent, the first targeting agent complement, 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 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.