Cross-reactivity in antibody microarrays and multiplexed sandwich assays: shedding light on the dark side of multiplexing.

Immunoassays are indispensable for research and clinical analysis, and following the emergence of the omics paradigm, multiplexing of immunoassays is more needed than ever. Cross-reactivity (CR) in multiplexed immunoassays has been unexpectedly difficult to mitigate, preventing scaling up of multiplexing, limiting assay performance, and resulting in inaccurate and even false results, and wrong conclusions. Here, we review CR and its consequences in single and dual antibody single-plex and multiplex assays. We establish a distinction between sample-driven and reagent-driven CR, and describe how it affects the performance of antibody microarrays. Next, we review and evaluate various platforms aimed at mitigating CR, including SOMAmers and protein fractionation-bead assays, as well as dual Ab methods including (i) conventional multiplex assays, (ii) proximity ligation assays, (iii) immuno-mass spectrometry, (iv) sequential multiplex analyte capture, (v) antibody colocalization microarrays and (vi) force discrimination assays.

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