Taguchi design-based optimization of sandwich immunoassay microarrays for detecting breast cancer biomarkers

Taguchi design, a statistics-based design of experiment method, is widely used for optimization of products and complex production processes in many different industries. However, its use for antibody microarray optimization has remained underappreciated. Here, we provide a brief explanation of Taguchi design and present its use for the optimization of antibody sandwich immunoassay microarray with five breast cancer biomarkers: CA15-3, CEA, HER2, MMP9, and uPA. Two successive optimization rounds with each 16 experimental trials were performed. We tested three factors (capture antibody, detection antibody, and analyte) at four different levels (concentrations) in the first round and seven factors (including buffer solution, streptavidin-Cy5 dye conjugate concentration, and incubation times for five assay steps) with two levels each in the second round; five two-factor interactions between selected pairs of factors were also tested. The optimal levels for each factor as measured by net assay signal increase were determined graphically, and the significance of each factor was analyzed statistically. The concentration of capture antibody, streptavidin-Cy5, and buffer composition were identified as the most significant factors for all assays; analyte incubation time and detection antibody concentration were significant only for MMP9 and CA15-3, respectively. Interactions between pairs of factors were identified, but were less influential compared with single factor effects. After Taguchi optimization, the assay sensitivity was improved between 7 and 68 times, depending on the analyte, reaching 640 fg/mL for uPA, and the maximal signal intensity increased between 1.8 and 3 times. These results suggest that Taguchi design is an efficient and useful approach for the rapid optimization of antibody microarrays.