The microtiter plates were coated with diluted capture mAb at 4°C. After blocking and washing steps, serial dilutions of the analytes were added to the wells, and the plates were incubated for 1 h at 37°C. Then, 100 μL of diluted HRP-labeled mAb was added to each well, followed by incubation for 1 h at 37°C. The solution was incubated for 15 min at 37°C before the enzymatic reaction was stopped with 50 μL of 2 M H2SO4. The OD value of each well was measured at 450 nm. These values were plotted against the analyte concentration on a logarithmic scale, and the generated sigmoidal curve was mathematically fitted to Eq. 1. The LOD and the detection range of the sandwich immunoassay were the same as those of the competitive immunoassay.