Establishment And Application Of IC-ELISA For Detection Of DBP In Food

Phthalate Esters(PAEs) are commonly used in plasticizer products, with world’s annual output up to millions of tons. In the early 21 st Century, PAEs went popular in plasticizer market for its advantages of characteristics and price, with the expenditure more than 85 % in plasticizer. In 2011, “Plasticizer Event” in Tai Wan has raised the security and healthy concern about PAEs generally. 6 kinds of PAEs were confirmed as “priority controlled environmental contamination” by Environmental Protection Agency(EPA, US A), including Di-butyl Phthalate(DBP). Domestic hygiene standard(GB 9685-2008) has performed since 2008, limited DBP in 0.3 mg/kg migrating from food. PAEs are semi-volatile, thermally stable, and non-polar compounds, which are of similar structures. The gas chromatography(GC) with a capillary column is commonly used as PAEs separation technology, while the mass spectrometry(MS) with high sensitivity is generally adopted in PAEs detection. Therefore, GC-MS combined method is applied in detecting PAEs according to GB. As with other methods reported, each detection method has their own limitations: the pricy equipment; the complicated pre-processing; the unbefitting detection limit, etc. Application of immunological detection could solve those problems effectively, and has lots of advantages, for instance: specific, efficient and sensitive; simple to handle; low cost and time-saving.In this study, DBP was selected as the research objec t. DBP-NH2 hapten was produced by chemical synthesis, then, coupled with carrier proteins(BSA and OVA) through diazotization reaction with the coupling ratio of 1:30 and 1:42, respectively. Polyclonal antibody(Pc Ab) against DBP- BS A was prepared afterwards. The titer of Pc Ab is above 2.56×105, and the concentration of purified Pc Ab is 18.72 mg/m L. After optimization of ELIS A reaction conditions, the standard curve with a linear range from 10 ng/m L to 38.7 μg/m L and a detection limit of 6.44 ng/m L was established. The cross-reactivities of anti-DBP m Ab to other ten phthalate esters were less than 1% except for BBP(41.7%). Therefore, this method is also reliable and suitable for monitoring BBP in food, with the linear range from 31.12 ng/m L to 72.63 μg/m L. The average recoveries of DBP from negative beverage and water were both between 86.8 % and 118.7 %, while recovery of negative oil was between 91.2 % and 126.7 %. Here, the ELIS A method on detecting DBP was successfully established and applied to real sample(sample size 100) analyses. The result shows that DBP residues ratios in waters, beverages and cooking oils are 40 %, 94 %, 100 %, respectively; and DBP residues amounts are 17.16 ng/m L, 26.48 ng/m L, 86.14 ng/m L respectively. It was indicated that DBP in all samples was not exceeded.