A sensitive heterogeneous biotin–streptavidin enzyme-linked immunosorbent assay for the determination of di-(2-ethylhexyl)phthalate (DEHP) in beverages using a specific polyclonal antibody

Abstract

Di-(2-ethylhexyl)phthalate (DEHP) is one of the long-chain or high-molecular-weight compounds of the phthalic acid diester (PAE) family, which is the most commonly used plasticizer and additive. However, DEHP may cause birth defects, sexual dysfunction, even cancers, possibly heart disease, etc. In order to detect DEHP with high sensitivity and specificity, an indirect competitive biotin–streptavidin enzyme-linked immunosorbent assay (BA-ELISA) has been established in this study. A specific polyclonal antibody (pAb-DEHP) targeting DEHP was obtained first, and the procedures of BA-ELISA were optimized for the determination of DEHP in beverages. Under optimal conditions, a good linearity was achieved within a range of 0.021 to 12.948 μg L⁻¹. The limit of detection (IC₁₀) was 0.0074 μg L⁻¹ and the median inhibitory concentration (IC₅₀) was 0.526 μg L⁻¹. The BA-ELISA was highly selective, with low cross-reactivity values with DEHP analogues (lower than 7%). Finally, the assay was successfully used to detect DEHP in beverages; the concentrations of DEHP in the samples ranged from 1.18 μg L⁻¹ to 40.68 μg L⁻¹. Satisfactory recoveries (89.07–109.33%) and coefficient of variation (CV) values (5.97% to 10.68%) were obtained, which further confirmed that this proposed BA-ELISA immunoassay is sensitive, rapid and accurate for monitoring DEHP in the environment.