Optimization of the ic ELISA

To improve the sensitivity of the ic ELISA, we optimized different parameters, including the antibody-working concentration, coating concentration, coating time, blocking solution, diluted concentration of GAMIgG-HRP, and time of TMB colorization. Concentrations of the working antibody and coating antigen were optimized by checkerboard titration using DES-MCPE-OVA as the coating antigen²⁵. Briefly, the DES-MCPE-OVA was coated on the ELISA plate at concentrations of 0.1, 0.2, 0.5, 1.0, 2.0, and 4.0 μg/mL and the mAbs towards DES were diluted with PBS at dilutions of 1:4.0 × 10³, 1:8.0 × 10³, 1:1.6 × 10⁴, 1:3.2 × 10⁴, 1:6.4 × 10⁴, 1:1.28 × 10⁵, 1:1.28 × 10⁵, and 1:5.12 × 10⁵. The OD_450nm value was obtained by microplate reader. A well was selected when its OD _{450 nm} value was approximately 1.0, and the difference of its OD_450nm value was significant relative to its adjacent wells. The concentrations of DES-MCPE-OVA and anti-DES mAbs corresponding to this well were considered optimal. For other parameters, the immunoassay performance was evaluated using the Amax (maximal absorbance)/IC₅₀ ratio. ELISA sensitivity is positively correlated with the Amax/IC₅₀ ratio²⁶.