*3.1. Antigen Design and Characterization*

In this study, quinoxaline haptens were designed with MQCA and DMEQ as the precursor substances and PABA and AOAA as the arms, respectively. The chemical structure of the precursor substances fully exposes the common structure of DQx and also carries reactive groups to facilitate the synthesis of hapten. Thus, the mAb prepared from such an immunogen could maximize the simultaneous recognition of multiple DQx. The haptens of MQCA–PABA and DMEQ–AOAA are small molecules that do not induce an immune response in the body and need to be conjugated with large proteins to be immunogenic. The prepared hapten was used as the basis for the synthesis of the conjugated product by the NHS active ester method. With the free carboxylic group, MQCA–PABA and DMEQ– AOAA were individually linked to proteins [21,22]. BSA and OVA, which served as the most common candidates of carrier protein, were adopted for the assay.

The spectra of the hapten, carrier protein, and conjugates were scanned using a UV– Vis spectrophotometer. The ultraviolet absorbance spectra of MQCA–PABA–BSA (λmax, 279 nm, 324 nm), MQCA–PABA–OVA (λmax, 280 nm, 322 nm), DMEQ–AOAA–HSA (λmax, 321 nm), and DMEQ–AOAA–OVA (λmax, 281 nm) were different from the carrier protein of the BSA (λmax, 279 nm) and OVA (λmax, 279 nm), and from the haptens of MQCA–PABA (λmax, 322 nm) and DMEQ–AOAA (λmax, 321 nm), which confirmed that the antigens were successfully synthesized (Figure 3). The estimated conjugation ratios of MQCA– PABA–BSA, MQCA–PABA–OVA, DMEQ–AOAA–HSA, and DMEQ–AOAA–OVA were 16.9, 10.7,17.8, and 11.3, respectively, obtained from the absorbance calculation analysis.

**Figure 3.** UV absorption peak spectra of 4 different antigens at 200–400 nm.
