Production Of Two Antibodies Against Phthalate Esters, The Establishment Of Enzyme-Linked Immunosorbent Assay And Application

Phthalate esters as a class of plasticizer are widely used, which comes mainly from industrial production and consumer markets. However, it is easy to dissociate and into the environment and food while in the production, use and processing,causing a large number of residues in the environment, pose a threat to human health,in order to investigate the distribution of these pollutants in the environment and assess the potential environmental hazards, thus it is necessary to establish an efficient and reliable method for the determination of trace PAEs in the environment,while, dibutyl phthalate and diethyl phthalate in the environment is generally detected two typical PAEs.At present, the detection of DBP and DEP mainly depends on the instrumental analysis methods including high performance liquid chromatography, liquid chromatograph-mass spectrometer and liquid chromatography-mass spectrometry etc,these methods have the advantages of accurate and quantitative, however, it is limited by the shortage of high cost, complex sample pretreatment and long testing cycle. In contrast, enzyme-linked immunosorbent assay(ELISA) which is based on antigen/antibody has been widely used in the environment and food safety testing for high-throughput, high specificity, fast and other advantages. Therefore, this research synthesized two hapten against DBP and DEP, corresponding antibodies were obtained by immunized animals, and develope ELISA which was based on the development of antibody for detection of dibutyl phthalate(DBP) and diethyl phthalate(DEP) in the environment and food, provide the basis for further investigation of contamination and risk assessment.The main contents are as follows:1. This study successfully prepared the hapten 4-amino-dibutyl phthalate(4-DBAP) and prepared immunogen DBAP-BSA via diazotization linkage. The DBAP-BSA conjugations were used as immunogen to immunize New Zealand white rabbits, then the polyclonal antibody against dibutyl phthalate(DBP) was successfully prepared, the titer of which was 25.6×104, and it cross-reacted with Benzyl butyl phthalate(BBP) and diethyl phthalate(DEP), its cross-reactivity rateswere 22.5% and 1.08%, respectively. A indirect competive ELISA for the detection of DBP was developed after optimizing, the 50% inhibition concentration(IC50) of the standard curve was 421 ng/m L, the linear detecting range were 70~1600ng/m L.,and the correlation coefficient R2 was 0.9918. The limit of detection(LOD) was 66.6ng/m L, and the recoveries were 79.7%~105.4% with coefficient of variation(CV) of3.25%~10.2%.2. This study successfully prepared the hapten 4-amino-diethyl phthalate(4-DEAP) and prepared immunogen DEAP-BSA via glutaraldehyde linkage. The DEAP-BSA conjugations were used as immunogen to immunize Balb/C mouse, then One cell line named 4G2 which could secrete monoclonal antibody was obtained,the titer of which was 9×104, and it cross-reacted with dibutyl phthalate(DBP) 、Monoethyl phthalate(MEP) and Benzyl butyl phthalate(BBP), its cross-reactivity rates were 4.29%、2.03% and 0.035%, respectively. A indirect competive ELISA for the detection of DEP was developed after optimizing, the 50% inhibition concentration(IC50) of the standard curve was 72 ng/m L, the linear detecting range were 20~320ng/m L., and the correlation coefficient R2 was 0.9916. The limit of detection(LOD) was 11.9 ng/m L, and the recoveries were 76.3%~103.6% with coefficient of variation(CV) of 2.67%~11.48%.3. We detected the concentraction of DBP and DEP in some small shallow river water, sediments and the common foods such as milk, coffee, wine, in Zhenjiang.The results showed that DBP and DEP in the urban rivers were more serious(except Yudai River due to regular dredging, thus the water quality is better), should be urgent treatmented. DBP and DEHP in food residues are as follows: ND~300.8ng/m L and ND~112.3 ng/m L, in which the content distribution dibutyl phthalate as follows: Wine > Coffee> Milk. Overall, the seized food two PAEs were lower than the national food safety standards.