Optimization of protein array fabrication for establishing high-throughput ultra-sensitive microarray assays for cancer research

The microarray format is well suited for measuring biomarkers since it is a sensitive multiplex assay that requires small amounts of material. Antibody-based arrays, referred to as forward phase assays, however, require extensive optimization, because these molecules have variable af nities and speci cities [4]. Typically, antibody speci city is determined by the Immuno (Western) blot method where a single band at the matched molecular weight is an indication of speci city. Antibody af nity is a more complicated parameter since it determines the linear range of the assay. In multiplex assays using antibodies with variable af nities, linearity would be dif cult to determine for every feature unless the readout can be carried out in solution over the course of multiple washing steps.