Artificial Antigen Synthesis and Antibody Production for Di(2-Ethylhexyl) Phthalate

A hapten di(2-ethylhexyl) 4-aminophthalate (DEHAP) which possesses the structural feature of di(2-ethylhexyl) phthalate (DEHP) was synthesized via esterification and reduction with 4-nitrophthalic acid and 2-ethyl-1-hexanol. The product was confirmed by infrared absorption spectroscopy (IR), ¹H nuclear magnetic resonance spectroscopy (¹H NMR) and mass spectrometry (MS). Then glutaraldehyde was used as crosslinker to link DEHAP and bovine serum albumin (BSA) to make artificial immunogen DEHAP-BSA. DEHAP was linked to ovalbumin (OVA) through diazotization to prepare coating antigen DEHAP-OVA. The structure of artificial antigens was identified by ultraviolet spectroscopy (UV) and SDS polyacrylamide gels electrophoresis (SDS-PAGE). BALB/c mice were immunized with DEHAP-BSA and the titer of antisera they produced were detected by indirect enzyme-linked immunosorbent assay (ELISA). Results showed that the artificial antigens for DEHP were synthesized successfully and specific polyclonal antibodies with high titer were produced, which made it possible to obtain monoclonal antibodies and establish immunoassay for DEHP.