Development of an enzyme-linked immunosorbent assay for podophyllotoxin

A competitive indirect enzyme-linked immunosorbent assay (ciELISA) for podophyllotoxin was developed by using polyclonal antibody, and its suitability for the determination of this analyte in spiked water samples was studied. To avoid antibody production to the linker, the succinoyl-podophyllotoxin (hapten) mimicking the analyte was synthesized and conjugated with the carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) by mixed anhydride reaction (MAR) and active ester method (AEM). Polyclonal antibodies raised against Hapten-BSA1 synthesized by MAR and Hapten-BSA2 by AEM were screened and selected for the ciELISA. One set of antibodies from the rabbit M4440 immunized with Hapten-BSA1 showed an I50 value of 2.21 μg/mL with a detection limit of 0.12 μg/mL, and the other set from the rabbit M4469 immunized with Hapten-BSA2 had an I50 value of 0.7897 μg/mL with a detection limit of 0.0056 μg/mL. This assay showed the cross-reactivities with the structurally closely related compounds. Recoveries from the podophyllotoxin-fortified tap water in the assay were in the range of 72-115%. A good correlation between podophyllotoxin concentration measured by the ELISA and HPLC (R2 = 0.9924, Y = 1.195X − 0.257) was obtained from linear regression analysis. These results indicate that the ELISA could be a convenient and supplemental analytical tool for monitoring podophyllotoxin and its analogues in waters without previous extraction or cleanup.