Capillary electrophoresis immunoassay.

397 In recent years, powerful, automatable methods have been developed for the analysis of DNA and transcribed RNA. In contrast, methods for analyzing translated proteins and their myriad post-translational modifications are largely fragmented, with no clearly dominant method. Further, automation of these methods is in its infancy. The most widely used method for analyzing proteins including their post-translational modifications is Western blotting, which remains little-changed in the ∼25 years since its first development. The amount of material required for a Western blot prevents analysis of small samples or single cells. Analysis of Western blots for different proteins and protein forms is cumbersome, limiting the level of multiplexing routinely achieved. We have developed a capillary-based immunoassay that is functionally equivalent to Western blotting while providing enormously better sensitivity, potentially down to the level of single cell analysis. It is equally amenable to discreet capillary or microchannel device formats. Importantly, it is inherently automatable, completely avoiding the gel transfer and electroblotting steps characteristic of Western blotting. In its most common form, the assay begins with isoelectric focusing (IEF) of proteins in a capillary or capillary channel. This is followed by immobilization of the separated proteins within the capillary lumen. Antibodies are then flowed through the lumen where they bind to their target proteins. In one form, primary antibodies are then bound by secondary antibodies with attached chemiluminescence reporter enzymes. Finally, chemiluminescent substrates are flowed through the capillary in a continuous stream, providing continuous chemiluminescence detection without exhaustion of the substrate.