Researches On Highly Sensitive Immunoassays For The Detection Of Dipropyl Phthalate And Dibutvl Phthalate In Foodstuff

The occurrence of Taiwan’s plasticizer food scandal captured theattention of consumers in2011. Phthalate esters (PAEs) are used in avariety of industrial and consumer applications, while PAEs can bereadily dispersed into the environment and foodstuff during the processesof production, use, or disposal. PAEs belong to endocrine disrupterchemical family, which can affect reproduction and development oforganisms by disturbing hormone synthesis; thus, there is necessary andsignificant to study PAEs. Recently immunoassay has the advantages ofhigh specificity, high sensitivity, and rapidity. In this paper, we developedthree immunoassay methods for ultra-trace analysis of DPrP and DBP infoodstuff and plastic food contact materials. The main content of thispaper are as follows:1. Two artificial antigens for DPrP were prepared by a diazotation method.Hapten DPrAP was covalently attached to BSA to be used as immunogen,which was applied to immunize New Zealand rabbits. The polyclonalantibodies were obtained when the titers were1:128,000. Thecross-reactivities of structurally related phthalate esters were below13%.Thus, the specific polyclonal antibodies were raised. Then the conjugatesof DPrAP and OVA were chosen as coating antigen, was used in thefollowing immunoassays.2. The conjugation of HRP with antigen (DPrAP-OVA) was prepared. Adirect competitive enzyme-linked immunosorbent assay (dc-ELISA) has been developed for the detection of DPrP in plastic food contact materialsbased on antibody-coating format. The absorbance at490nm wasmeasured using a microplate reader. Under the optimal conditions, thelimit of detection (LOD) of the method was0.01ng/mL, and a goodquantitative working range of0.01-16ng/mL was obtained. Therecoveries of DPrP in four real samples ranged from85.9%to109.4%.3. The DPrAP-OVA were coupled to HRP as detectable probe. A directcompetitive chemiluminescent enzyme-linked immunosorbent assay(CL-ELISA) based on polyclonal antibodies with enhancedchemiluminescent for the detection of DPrP in foodstuff has beendeveloped. Chemiluminescent intensity (ICL) was monitored on amicroplate reader. Under the optimal conditions, the LOD of the methodwas0.03ng/mL, and a good quantitative working range of0.03-500ng/mL was obtained. The recoveries of DPrP in six real samples rangedfrom86.4%to119.5%.4. A new fluorescein of BODIPY was coupled to antibody. A directcompetitive fluorescence immunoassay (dc-FIA) based on polyclonalantibodies for the detection of DBP in food samples has been developed.Fluorescence intensity was monitored on a microplate reader at λex=485nm, λem=528nm. Under the optimal conditions, the LOD of the methodwas0.001ng/mL, and a good quantitative working range of5.7×10-3-1.1×103ng/mL was obtained. The recoveries of DPrP in threereal samples ranged from87.0%to105.0%.