Antibody-based arrays for detecting multiple signal transducers in rare circulating cells

An assay for performing a multiplex, high-throughput immunoassay having superior dynamic range, the assay comprising:

• (a) incubating a cellular extract with a plurality of dilution series of capture antibodies specific for one or more analytes in the cellular extract to form a plurality of captured analytes, wherein the capture antibodies are restrained on a solid support;
• (b) incubating the plurality of captured analytes with detection antibodies specific for the corresponding analytes to form a plurality of detectable captured analytes, wherein the detection antibodies comprise:
• (1) a plurality of activation state-independent antibodies labeled with glucose oxidase, and
• (2) a plurality of activation state-dependent antibodies labeled with a first member of a signal amplification pair, wherein the first member of the signal amplification pair is a peroxidase,
• wherein glucose oxidase generates hydrogen peroxide (H2O2), which channels to and reacts with the first member of the signal amplification pair;
• (c) incubating the plurality of detectable captured analytes with a second member of the signal amplification pair to generate an amplified signal;
• and
• (d) detecting the amplified signal generated from the first and second members of the signal amplification pair.