*2.4. Development of ic-ELISA Analysis*

First, the purified antibody was titrated with different coating antigens in a square matrix to find the best concentration so that the sensitivity of the ic-ELISA assay would be the highest [19]. Then, the gradient standard solutions of DMEQ (0.8, 1.6, 3.2, 6.4, and 12.8 μg/L) were analyzed by ic-ELISA using the optimal concentrations optimized as above. The absorbance values obtained (OD450nm) were plotted against the corresponding concentrations in a standard calibration curve, and the concentration of DMEQ that produced half inhibition (IC50 value) was obtained from the results.

The extent of cross-reactivity (CR) of this mAb to different drugs was calculated by measuring the IC50 values of different structural analogs. Five DQx (DMEQ, DOLA, DQCT, DCYX, and DCBX) and six structural analogs (N1-DCYX, N4-DCYX, MQCA, MQCA– PABA, MEQ, and QCA) were used to determine the CR of the mAb. The IC50 values of all compounds were determined from the measured OD450nm with the standard curve, and based on the IC50 values, the CR was calculated as follows:

CR (%) = [IC50 (standard drug)/IC50 (analogues)] × 100%
