Combining positive attributes of planar arrays and suspension arrays, immobilized particle arrays offer a new format in which immobilized submicrometer particles are arrayed on hydrogel-coated slides, providing 100+ assay replicates within each spot. This research describes how to prepare immobilized protein arrays and how to assay the binding of labeled target molecules to the arrayed capture probes. The assay system exhibits an intrinsic dynamic range of two to three decades, with coefficients of variation from 5 to 10%. For antibody-antigen binding, target capture appears to be reaction rate limited. For labeled antibody binding to antigen on the immobilized particles, the detection limit is approximately 0.5 ng/mL. When antibodies on the immobilized particles exhibit multivalent binding of target molecules, the detection limit is approximately 0.01 ng/mL. For protein arrays, potential advantages of this format are improved coating of the capture reagent, an increased number of options for protein presentation, reduced mass transport effects, and higher density multiplexing.
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