Synthesis of an artificial antigen and preparation of a polyclonal antibody for the sensitive determination of phthalate esters by enzyme-linked immunoassay

Abstract

In this study, two generic haptens of phthalate esters (PAEs) were synthesized by introducing spacer arms with different lengths at the meta-position of an ester group of dimethyl phthalate (DMP). Immune and coating antigens were prepared by coupling the haptens with bovine serum albumin (BSA) and ovalbumin (OVA), respectively. Polyclonal antibodies against two immunogens were obtained, and their sensitivities and specificities were examined by enzyme-linked immunosorbent assay (ELISA). Under the optimal experimental conditions, the IC₅₀ values of eight PAEs were obtained in the range of 17.12–102.57 ng mL⁻¹ with the limits of detection (LODs) around 0.012–0.042 ng mL⁻¹. The prepared polyclonal antibody with a higher titer displayed a broad cross-reactivity in the range of 16.69–83.84% for the other seven PAEs (except DMP). The average recoveries of PAEs at the spiked levels of 0.5, 1.0 and 10.0 ng mL⁻¹ ranged from 63.9% to 103.6% with relative standard deviations (RSDs) below 11.32%, which was consistent with gas chromatography-mass spectrometry (GC-MS). The developed ELISA method was successfully applied to determine multiple PAEs in greenhouse soils.