Applications demonstrating protein microarray utility

Assays utilized conventional ELISA processing except that the reagent volumes were greatly reduced, ranging from 25- to 50-pL well additions. Following a 1-hr block in casein, the monoclonal detection antibody was incubated an additional hour at room temperature. The assay sensihvity from the micro-ELISA was approximately 13.4 ng/mL for rabbit IgG — a result similar to that reported by Silzel et al. (1998). The benefit of the array of arrays approach is that 96 samples could be processed for multiple (36 to 144) analytes within the same time interval as a standard single-analyte ELISA. 

Conventional plastic microtiter plates have also been adopted for use with protein microarrays in the array of arrays format. Matson et al. (Oak Ridge 

Perhaps the first published demonstration of high density applications for protein microarrays came from the work of MacBeath and Schreiber at Harvard (2000). Proteins were arrayed onto aldehyde-activated glass slides and analyzed in much the same manner described for the creation of cDNA microarrays (Schena et al., 1995), including the use of dual-color label 

The immobilization strategies are of particular interest. The authors reasoned that the use of aldehydes to tether proteins to the solid phase could be ideal for certain protein-protein interaction studies. Since many protein-lysine residues are available for coupling to aldehydes via Schiff s base, a number of spatial orientations are possible. Such random oriented attachments would permit exposure of various surfaces of a protein to the solution, and new protein-protein interactions would be potentially possible. 

Another useful strategy is scaffolding. For example, immunoassays employ BSA both as a blocking agent to reduce nonspecific adsorption of other proteins and also as a scaffold. Essentially, BSA is first attached to the solid support and then further derivatized for the coupling of additional capture ligands. MacBeath and Schreiber first formed a monolayer of BSA and then printed proteins on top of the monolayer. In this manner, small proteins were expressed on the surface and not buried by the BSA. 

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