Sigmoidal plots are shown to quantify the limit of detection (>100 pg/mL) for EpCAM protein using the monoplexed approach compared to the limit of detection (>103pg/mL) assessed by a traditional colorimetric sELISA assay with absorbance read-out. All experiments were performed in triplicates (N=3, >10,000 microspheres per condition were analyzed by imaging).