2.2.2. Synthesis of Antigens DMEQ–AOAA–HSA/OVA

As shown in Figure 2B, AOAA (130.0 mg) was added to 6 mL of pyridine containing DMEQ (200.0 mg) and reacted at 60 ◦C for 6–8 h. Then, the pyridine was removed with a rotary evaporator, and 20 mL of saturated sodium bicarbonate solution and 50 mL of ethyl acetate were added. After shaking well, the aqueous layer was adjusted to pH 3 with hydrochloric acid and extracted twice with ethyl acetate. Finally, it was dried with anhydrous magnesium sulfate, and the filtrate was evaporated after filtration to obtain the solid: DMEQ–AOAA. The hapten was conjugated to the carrier protein in the same way as above, except that BSA was replaced with HSA. The haptens, carrier proteins, and conjugates were scanned under a UV spectrophotometer (200–400 nm) to determine the state of conjugation and to record the corresponding maximum absorption wavelength and absorption values. The relevant absorbance coefficient (K) was calculated from K = A/CL, and the conjugation ratio was calculated from [K(conjugation) − K(carrier protein)]/K(hapten).
