Quantitative protein profiling using antibody arrays

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Abstract
Traditional approaches to microarrays rely on direct binding assays where the extent of hybrid‐isation and the signal detected are a measure of the analyte concentration in the experimental sample. This approach, directly imported from the nucleic acid field, may fail if applied to antibody‐antigen interactions due to the shortage of characterised antibodies, the significant heterogeneity of antibody affinities, their dependence on the extent of protein modification during labelling and the inherent antibody cross‐reactivity. These problems can potentially limit the multiplexing capabilities of protein affinity assays and in many cases rule out quantitative protein profiling using antibody microarrays. A number of approaches aimed at achieving quantitative protein profiling in a multiplex format have been reported recently. Of those reported, the three most promising routes include signal amplification, multicolour detection and competitive displacement approaches to multiplex affinity assays. One in particular, competitive displacement, also overcomes the problems associated with quantitation of affinity interactions and provides the most generic approach to highly parallel affinity assays, including antibody arrays.