Synthesis and identification of artificial antigen for dibutyl phthalate

Objective This study was aimed to prepare and identify a new kind of artificial antigen for dibutyl phthalate.

Method Dibutyl 4-aminophthalate (DBAP) was obtained via esterification and reduction of 4-nitrophthalic acid. The hapten derivative monoglutaryl 4-amino-dibutyl phthalate (DBCP) was prepared through DBAP reacting with glutaric anhydride. DBCP was conjugated to bovine serum albumin (BSA) or ovalbumin (OVA) to make artificial immunogen via activated ester method. Then the antiserum of two female New Zealand white rabbits was obtained after injecting with artificial immunogens which were successfully coupled. Indirect competitive enzyme-linked immunosorbent assay (icELISA) was used to analyze the sensitivity and specificity of the antiserum.

Result The successful synthesis of hapten derivative was identified by mass spectrometry (MS) and ¹H nuclear magnetic resonance (¹H NMR). The successful coupling was proved by ultraviolet spectroscopy (UV) and SDS polyacrylamide gels electrophoresis (SDS-PAGE). Furthermore, the numbers of DBCP molecules per BSA/OVA molecules for two conjugates were 11:1 and 17:1 respectively, estimated by ultraviolet spectrophotometry. The icELISA method indicated high specificity for the antiserum with the highest titer being 1:128 000 for samples from both rabbits, and the IC₅₀ being 301 and 308 ng·mL^-1 respectively.

Conclusion The hapten derivative can be synthesized in a convenient and secure way. The successful synthesis of the artificial antigen for dibutyl phthalate makes it possible to get polyclonal antibodies and establish immunoassay for DBP.