Direct competitive immunosorbent assay for detection of MEHP in human urine

Abstract Di-(2-ethylhexyl) phthalate (DEHP) is the most commonly used plasticizer for flexible polyvinyl chloride (PVC), which is also known as one of the environmental endocrine disruptors with the reproductive, developmental and embryonic toxicity after entering human body. Mono-2-ethylhexyl phthalate (MEHP) is one of the most complicate metabolites from DEHP in vivo and responsible for many toxic effects of DEHP. In order to evaluate human exposure to DEHP, a direct competitive enzyme-linked immunosorbent (dcELISA) based on monoclonal antibody (mAb) was developed to detect MEHP. A hybridoma cell line 4B9 secreting mAb against MEHP was prepared, and the horseradish peroxidase (HRP) labeled antigen as a probe in the dcELISA was made. After optimization of ELISA reaction conditions, the standard curve with a linear range from 0.56 to 1000 ng mL −1 and a detection limit of 0.39 ng mL −1 was established. The cross-reactivities of anti-MEHP mAb to other ten phthalate esters were less than 5% except for mono-methylphthalate (MME). The average recoveries of MEHP from distilled water and negative human urine were both between 87.4% and 94.72% with coefficient of variation (CV) less than 5%. Here, the ELISA method on detecting MEHP was successfully established and applied to real urine sample analyses and the results were confirmed by HPLC. Furthermore, it was indicated that the immunoassay was reliable and suitable for monitoring MEHP.