Characterization and optimization of PEdELISA assay. PEdELISA standard curves for the 4 cytokines: IL-6 (A), MCP-1 (B), TNF-α (C), and IL-2 (D). The step 1 incubation time varied from 60 to 300 seconds, and the step 2 incubation time was fixed at 30 seconds. Due to the extreme underlabeled nature of the assay, the 15- and 30-second assays were performed by merging the step 1 and step 2 process into a single step by mixing all required reagents. The LOD was determined by concentration from the reagent blank’s signal + 3σ (dotted line). Correlation between PEdELISA and conventional sandwich ELISA tests for the 4 cytokines using spike-in recombinant proteins in 25% fetal bovine serum: (E) 15-second PEdELISA incubation time (R² = 0.92); and (F) 300-second PEdELISA incubation time (R² = 0.97). The ground truth is plotted with a dotted line with scattered predetermined spike-in concentrations. (G) Theoretical (line) and experimental (scatter) LOD of PEdELISA as a function of the step 1 incubation time for 4 cytokines.