*2.2. Preparation of Monoclonal Antibody*

The 1H2 cells reached logarithmic phase four days after resuscitation. Ascites were collected 10 days after injection. The purification effect of the monoclonal antibody was determined with SDS-PAGE (12% separation gel, 5% spacer gel). The purified immunoglobulin G antibody has only two main bands, heavy chain and light chain, indicating that the purification method can remove the heteroprotein in ascites. The relationship between the molecular weight of the protein marker and the mobility of the heavy and light chains was obtained. The molecular weights of heavy and light chains were about 50 and 25 kDa, respectively (Figure 1). The purified antibodies proved to be capable of meeting the requirements for the next experiment.

**Figure 1.** Purification and identification of antibody by SDS-PAGE electrophoretogram.

#### *2.3. Synthesis of the OTA-FL Tracer*

The carboxyl groups in OTA are inactive; for their activation, we used N, N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide in an aprotic solvent medium. In this research, four typical kinds of dyes with different Ex/Em wavelengths (HDF, BDF, AMF, and EDF) were selected for covalent conjugation to OTA.

After preliminary purification through thin layer chromatography (TLC), principal bands of OTA-AMF, OTA-BDF, OTA-EDF, and OTA-HDF were collected. The molecular weights of OTA-AMF, OTA-BDF, OTA-EDF, and OTA-HDF were 747.15, 863.33, 837.29, and 891.38, respectively (Figure 2). The mass spectra [M+] ion peaks were 747.45, 863.45, 835.45, and 891.50, respectively, which are consistent with the molecular weights of the target compounds (Figure 3). Dye-labelled tracers were primarily designed to bind to the specific monoclonal antibodies to determine whether the OTA-AMF, OTA-BDF, OTA-EDF, and OTA-HDF could provide satisfactory results. All the tracers induced a significant rise in FP signals before and after the addition of saturated quantities of mAbs (Figure 4). The δFP values of the tracers ranged from 5 to 207 mP, which are adequate for application in FPIA reaction progress monitoring. This proved that the dyes were successfully conjugated to the corresponding mycotoxin [18]. To improve the detection sensitivity, the OTA-AMF that had the highest δFP value was chosen for further optimization.

**Figure 2.** Structural formulas of tracer OTA-AMF, OTA-BDF, OTA-EDF, and OTA-HDF.

**Figure 3.** Mass spectra of (**a**) OTA-AMF, (**b**) OTA-BDF, (**c**) OTA-EDF, and (**d**) OTA-HDF.

**Figure 4.** The result of four tracers combined with the diluted specific monoclonal antibodies (*n* = 3).
