*3.2. Characterization of mAb*

The immunogens MQCA–PABA–BSA and DMEQ–AOAA–HSA were inoculated into mice to produce mAb. After the third injection, the specificity and titer of the immunized mouse antisera were determined using homologous and heterologous coatings, respectively (Table 1). The analysis in the table shows that DMEQ–AOAA–HSA showed poor specificity as an immunogen, both in homologous and heterologous coating. The immunogen MQCA– PABA–BSA with the coating antigen MQCA–PABA–OVA showed high titers but poor specificity against DMEQ. However, DMEQ–AOAA–OVA showed a better inhibition rate as a coating antigen for heterologous analysis. Therefore, splenocytes from mice immunized with MQCA–PABA–BSA were fused with myeloma cells. In the process of preparing an antibody, we similarly found that the heterologous coating antigen DMEQ– AOAA–OVA was more suitable for ic-ELISA than the homologous coating antigen MQCA– PABA–OVA. After multiple cell subcloning, a single strain of hybridoma cells 1A3 showed better recognition of DMEQ. Therefore, this cell line was selected for the production of monoclonal antibodies.


**Table 1.** The titer and specificity of the antiserum.

<sup>1</sup> B is the absorbance value of DMEQ addition, and B0 is the absorbance value of PBS addition of equal amount.

First, the obtained antibodies were optimized for the ic-ELISA conditions. The results showed that the ratio of 1:2000 mAb and 6.0 μg/mL of the coating antigen (DMEQ–AOAA– OVA) worked best, and the rest of the experiments were performed under this condition. As shown in Figure 4, the standard curve was fitted with the logarithmic value of DMEQ concentration as the *X*-axis and B/B0 as the *Y*-axis. The standard curve ranged from 0.8–12.8 μg/L, and the IC50 value was 2.84 μg/L based on the DMEQ standard solution. In Table 2, it can be seen that the CR of mAb showed varying levels of cross-reactivity to MQCA–PABA (44%), DOLA (27%), MQCA (2%), DQCT (1.3%), and QCA (1%) but showed no measurable CR (CR < 0.1%) with DCBX, DCYX, N1-DCYX, N4-DCYX, and MEQ.

**Figure 4.** The standard curve of ic-ELISA.


**Table 2.** The cross-reactivity of mAb to different analytes.
