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Research on the Immunoassays for the Determination of Environmental Hormones: Diethyl Phthalate and Dicyclohexyl Phthalate

Author: ShengYaLi
Tutor: ZhangMingCui
School: Anhui Normal University
Course: Analytical Chemistry
Keywords: diethyl phthalate dicyclohexyl phthalate environmental hormone antigen antibody enzyme-linked immunosorbent assay fluorescence immunoassay
CLC: O657.3
Type: Master's thesis
Year: 2010
Downloads: 104
Quote: 0
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Abstract

Phthalate esters （PAEs） are widely used in the manufacturing and processing of plastic products as plasticizers and they are easily bioaccumulated in the aquatic and the terrestrial environment become one of the most universal pollutants existing in atmosphere, water, soil, organism and so on widely. PAEs, one kind of important endocrine disrupting chemicals （EDCs） can enter into animal’s bodies and produce similar effect of estrogen. They will prevent the normal hormones which are related with animal procreation and growth from synthesizing, secreting, storing, transporting, combining and eliminating, and interfere in hormones keeping balance in blood so as to influence the procreation and growth of animals. Some of PAEs are considered to be potential carcinogens, teratogens and mutagens.The investigation of literature shows that immunoassay is seldom used in monitoring environmental hormone, which means further research is promising and necessary. The purpose of this paper is to build a new immunoassay method for detecting environmental hormone phthalate esters. For this aim, some researches were developed as follows. 1. Diethyl 4-amino phthalate （4-DEAP） and dicyclohexyl 4-amino phthalate （4-DCHAP） as hapten derivatives were synthesized in our previous work. Then DEAP and DCHAP were conjugated to BSA via amino diazotization linkage. The DEAP-BSA and DCHAP-BSA conjugations were used as immunogen to immunize female New Zealand white rabbits. The antiserum with a sufficiently high titer was generated in rabbits. The double immunodiffusion test and an indirect fluorescence immunoassay were developed for titration of the sera titer. The final sera from rabbits had titers with dilutions of 1:32 by using double immunodiffusion test and 1:25600 by using indirect fluorescence immunoassay, respectively. Then the animals were exsanguinated and the blood was collected and centrifugated, the IgG fraction of the antiserum was isolated by precipitation with saturated ammonium sulfate solution. After dialysis against PBS, the purified IgG fractions were stored at -20°C until use.2. A sensitive and specific indirect competitive enzyme-linked immunosorbent assay （ELISA） and a fluorescence immunoassay （FIA） have been developed for the detection of dicyclohexyl phthalate （DCHP） using an antigen-coated plate format. The first assay ELISA had a practical working concentration range from 0.2 to 100μg L^-1 and the limit of detection was 0.02μg L^-1. Other similar phthalate compounds do not interfere obviously and the cross-reactivity rates were less than 10%. Fluorescence immunoanalytical technique of dicyclohexyl phthalate was established with the signal of fluorescein isothiocyanate. Under optimized FIA condition, the quantitative working range was 0.1-200μg L^-1 with a limit of detection of 0.05μg L^-1,and the cross-reactivity rates were less than 10%. Four kinds of water samples （tap water, lake water, river water and leachate from drinking bottles） had been detected. The result showed that ELISA and FIA procedures can be used to detect dicyclohexyl phthalate in environmental samples.3. An indirect competitive ELISA for the diethyl phthalate using an antibody-coated plate format has been developed. The quantitative working range was 0.2-400μg L^-1 with a limit of detection of 0.08μg L^-1, and the cross-reactivity rates were less than 10%. Each water sample （tap water, lake water, river water and leachate） was fortified with DEP at several concentration levels and were directly analyzed with only dilution with an equal volume of antiserum solution. The proposed ELISA turned out to be a powerful tool for monitoring of diethyl phthalate in water samples.

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