Abstract: AimTo synthesize and identify artificial antigen of podophyllotoxin for the production of podophyllotoxin polyclonal antibody. MethodsThe hapten was synthesized by two different chemical approaches and characterized by TLC, IR, NMR, and MS. Mixed anhydride reaction (MAR) and active ester method (AEM) were used to couple the podophyllotoxin to carrier proteins (BSA and OVA). Characterization of artificial antigens was done by using spectroscopy and electrophoresis. The anti-podophyllotoxin polyclonal antibodies were obtained through immunizing rabbits. ResultsThe results from IR, NMR and MS showed that 4-O-succinoyl podophyllotoxin (hapten) was successfully synthesized. The coupling molar ratios of the hapten and carrier proteins were 88.6 for Hapten-BSA₁, 40.3 for Hapten-BSA₂, 17.8 for Hapten-OVA₁, and 54.2 for Hapten-OVA₂. Hapten conjugates coupled with BSA yielded two sets of the specific and affinitive polyclonal antibodies. One set of antibodies showed an IC₅₀ value of 2.21 μg·mL^-1 with a detection limit of 0.12 μg·mL^-1. ConclusionAntigenic conjugates were artificially synthesized, and based on these artificial antigens, polyclonal antibodies against podophyllotoxin were raised from rabbits immunized with two different immunogens and characterized with an indirect ELISA format.