Abstract:

Four hapten derivatives were obtained from 4-nitrophthalic acid through esterification and reduction. In order to
prepare artificial antigens, the hapten derivatives were coupled with the carrier proteins bovine serum albumin (bovine serum
albumin, BSA) and ovalbumin (OVA) by diazotization method and activated ester method respectively. The successful
synthesis of artificial antigens was identified by ultraviolet spectroscopy (UV) and sodium dodecyl-sulfate polyacrylamide
gel electroporesis (SDS-PAGE). New Zealand white rabbits were injected with each of the four artificial antigens coupled
with BSA. The sensitivity and specificity of antibodies were determined by enzyme-linked immunosorbent assay (ELISA).
The results showed that the titer of DBAP-BSA (dibutyl 4-aminophthalate-BSA) antibody was 1:64 000 and the titers of
all three other antibodies were 1:128 000. The IC50 values of DBAP-BSA, DBCP-BSA (dibutyl 4-carboxylphthalte-BSA),
DiBAP-BSA (diisobutyl 4-aminophthalate-BSA) and DCHAP-BSA (dicyclohexyl 4-aminophthalate-BSA) against DBP
(dibutyl phthalate), DBP, DiBP and DCHP were 0.305, 0.268, 0.104 and 0.247 mg/mL, respectively. The cross-reactivity
with each other was small. Therefore, the antibodies had high sensitivity and specificity. The successful synthesis of four
hapten derivatives made it possible to prepare polyclonal antibodies and establish immunoassay.

Key words: phthalic acid ester, artificial antigen, antibody