Precise Chip-to-Chip Reagent Transfer for Cross-Reactivity-Free Multiplex Sandwich Immunoassays

Common multiplex sandwich immunoassays suffer from cross-reactivity due to the mixing of detection antibodies and the combinatorial, undesired interaction between all reagents and analytes. Here we present the snap chip to perform antibody colocalization microarrays that eliminates undesirable interactions by running an array of singleplex assays realized by sequestering detection antibodies in individual nanodroplets. When detecting proteins in biological fluids, the absence of cross-reactivity allows a higher level of multiplexing, reduced background, increased sensitivity, and ensures accurate and specific results. The use of the snap chip is illustrated by measuring highly related analytes such as proteins isoforms and phospho-proteins, both particularly prone to cross-reactivity, in a single experiment. The main steps of the protocol are preparation of sample, incubation on an assay slide harboring the microarrayed capture antibodies, transfer of the microarrayed detection antibodies on their cognate spots, and measurement of the assay results by fluorescence.