*3.1. Antigen Verification and Serum Antibody Monitoring*

The design and synthesis of antigens are very important for promoting the immune response of mice and preparing mAb with high-affinity and high-sensitivity. PYR is a small molecule compound that lacks functional groups conjugated with proteins and has no immunogenicity. In this study, pyrene butyric acid (PBA) was used as a hapten to synthesize artificial complete antigen. PAHs are composed of multiple benzene rings and have unique UV absorption spectra, so PBA hapten has multiple absorption peaks. The UV scanning spectra of immunogen PBA-BSA and coating antigen PBA-OVA are shown in Figures 1 and 2.

**Figure 1.** UV spectrum of BSA, PBA and PBA-BSA.

**Figure 2.** UV spectrum of OVA, PBA and PBA-OVA.

As shown in Figures 1 and 2, the UV absorption spectra of antigens changed significantly compared with those of carrier proteins (BSA, OVA) and PBA haptens and showed the characteristics of the absorption peaks of carrier proteins and haptens. The results showed that the PBA-BSA/OVA antigens were successfully synthesized. The estimated hapten/protein ratios of antigens were 8.1 (A1), 12.8 (A2), 18.3 (A3), 9.4 (B1), 11.7 (B2), and 19.2 (B3).

It has been reported that the immune effect of immunogens would be affected by the coupling rate of hapten and carrier proteins [27]. Therefore, we prepared antigens with different coupling ratios to explore their effects on the affinity and sensitivity of immunized mice. Three immunogens (A1, A2 and A3) were used to immunize mice. The absorbance values and inhibition rates of antiserum under different coupling ratio antigens are shown in Table S1. It can be seen that in a certain range, the higher the hapten/protein ratio was, the better the serum titre and affinity of the antiserum were. This may be due to more haptens in the conjugate being exposed, which stimulated the mouse to produce more antibodies against PYR. Therefore, immunogen A3 and coating antigen B3 were the best combination.
