| "Environmental hormones" is a term given to endocrine disruptors, which are chemical substances capable of interfering with animal hormones, hence, it adversely affects the endocrine system and consequently alters the development and/or reproduction of organisms, including humans. Phthalic acid esters (phthalate esters, PAEs) are an important kind of environmental hormone. They are used most commonly as plasticizers in the food and construction industry, Consistent experimental evidence shows that some phthalates are developmental and reproductive toxicants in animals.In recent years, plasticizers have caused a wide distribution of phthalic acid esters in environment. PAEs become one of the most prevalent environmental contaminants, existing in atmosphere, water, soil, organism and so on widely, and have been measured in the majority of the general population.Gas chromatography, high performance liquid chromatography, mass spectroscopy, and gas chromatography-mass spectrometry are the usual methods for determination of PAEs, but they are time-consuming and have the high cost of instrumentation. Presently, there is a need for acceptable, rapid, reliable, sensitive, and cost-effective assay for determining the presence of phthalates. Indeed, immunoassay is a fast, cost-effective and reliable method for identifying and quantitating the presence of a target compound in samples. It can meet these requirements. But only a few references report the use of immunoassay techniques to measure phthalate esters quantitatively.The goal of this paper was to find some immunoassays to determine the presence of PAEs of environmental hormone. In this work, 5 kinds of PAEs environmental hormone, such as dimethyl phthalate, diethyl phthalate, dipropyl phthalate, dibutyl o-phthalate, dicyclohexyl phthalate, have been studied. The main efforts are concerned on the design and preparation of optimum haptens as immunogens and competitors, preparation and characterization of artificial antigen and antibody, and development of a series of immunoassays to detect PAEs in environmental samples. The specific procedures and results were as follows.(1) By virtue of 4-nitrophthalic acid as precursor compound, a new compound dipropyl phthalate hapten (4-amino dipropyl phthalate) has been synthesized through esterified reaction with n-propalol and then being reduced. It was introduced amino as a substituent on the aromatic ring and retained the ester group. The structure was characterized by ~1HNMR, IR and UV.(2) 4 kinds of phthalate haptens, such as 4-amino dimethyl phthalate, 4-amino diethyl phthalate, 4-amino dibutyl o-phthalate, 4-amino dicyclohexyl phthalate, have been prepared through new synthesized and purified methods. They were introduced amino as substituent on the aromatic ring and retained the ester group. The structures of all obtained compounds were characterized by ~1HNMR, IR and UV.(3) 5 kinds of phthalate haptens were conjugated to BSA via amino diazotization linkage. Thus 5 kinds of new artificial antigens were prepared and tested by fluorescence, reaction solution color. The products were used as an immunogen, demonstrating that it is suitable for polyclonal antibody production.(4) The conjugates were used as an immunogen. 5 kinds of new high titer polyclonal antibodies against phthalate esters were produced from New Zealand white rabbits immunized with the immunogen, the animals were exsanguinated and antiserum were purified.(5) Using the purified IgG, 9 kinds of novel fluorescence immunoassay for the determination of phthalate esters in environmental samples were assessed, and they were proved to be simple, rapid, sensitive and specific methods. Results were as follows.1) A novel antigen coated competitive fluorescence immunoassay with the equilibrium method for detection of diethyl phthalate (DEP) was described. Under best conditions, DEP can be determined in the concentration range of 6.0～60μg/L with a detection limit of 1.0μg/L. The regression equation of this assay is△F= 24180.1-1144.9logC(μg/L) and coefficient of correlation R~2 is 0.997. DEP can also be determined in the concentration range of 60～1000μg/L. he regression equation of this assay is△F= 38801.6-9230.9logC(μg/L) and coefficient of correlation R~2 is 0.998. Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique and the cross reactivity rates were less than 7%. The accuracy of the assay has been evaluated using tap water, water samples from Suzhou River (Shanghai, China), drinking water, water leaching from disposable plastic products and spiked samples, recovery rates were 89.8%～109.9%,and RSD were less than 12%.2) A novel antibody coated competitive fluorescence immunoassay with the equilibrium method for detection of diethyl phthalate was described. Under best conditions, DEP can be determined in the concentration range of 1.0～500μg/L with a detection limit of 1.0μg/L. The regression equation of this assay is△F=11024.9-693.3logC(μg/L) and coefficient of correlation R~2 is 0.996. Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique and the cross reactivity rates were less than 10%. The accuracy of the assay has been evaluated using tap water, water samples from Suzhou River (Shanghai, China), drinking water, water leaching from disposable plastic products and spiked samples, recovery rates were 90.2%～109.1%,and RSD were less than 14%.3) A novel antibody coated competitive fluorescence immunoassay for detection of dimethyl phthalate (DMP) was described. Under best conditions, DMP can be determined in the concentration range of 0.1～300μg/L with a detection limit of 0.1μg/L. The regression equation of this assay is△F=10381.3-658.9logC(μg/L) and coefficient of correlation R~2 is 0.998. Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique and the cross reactivity rates were less than 10%. The accuracy of the assay has been evaluated using tap water, water samples from Suzhou River(Shanghai, China), drinking water, water leaching from disposable plastic products and spiked samples, recovery rates were 92.4 %～108.2%,and RSD were less than 13%.4) A novel antibody coated competitive fluorescence immunoassay with the equilibrium method for detection of dipropyl phthalate (DPrP) was described. Under best conditions, DPrP can be determined in the concentration range of 1.0～200μg/L with a detection limit of 1.0μg/L. The regression equation of this assay is△F=6283.9-976.8logC(μg/L) and coefficient of correlation R~2 is 0.996. Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique and the cross reactivity rates were less than 9%. The accuracy of the assay has been evaluated using tap water, water samples from Suzhou River(Shanghai, China), drinking water, water leaching from disposable plastic products and spiked samples, recovery rates were 91.8%～109.4%,and RSD were less than 13%.5) A novel antigen coated competitive fluorescence immunoassay with the equilibrium method for detection of dibutyl o-phthalate (DBP) was described. Under best conditions, DBP can be determined in the concentration range of 1.0～100μg/L with a detection limit of 0.05μg/L. The regression equation of this assay is△F=4881.7-193.0logC(μg/L) and coefficient of correlation R~2 is 0.998. Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique and the cross reactivity rates were less than 9 %. The accuracy of the assay has been evaluated using tap water, water samples from Suzhou River (Shanghai, China), drinking water, water leaching from disposable plastic products and spiked samples, recovery rates were 90.5 %～107.5 % ,and RSD were less than 13 %.6) A novel antibody coated competitive fluorescence immunoassay with the equilibrium method for detection of dibutyl o-phthalate was described. Under best conditions, DBP can be determined in the concentration range of 0.5～400μg/L with a detection limit of 0.3μg/L. The regression equation of this assay is△F=6548.1-547.1logC (μg/L) and coefficient of correlation R~2 is 0.997. Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique and the cross reactivity rates were less than 9%. The accuracy of the assay has been evaluated using tap water, water samples from Suzhou River (Shanghai, China), drinking water, water leaching from disposable plastic products and spiked samples, recovery rates were 90.1 %～106.7 %,and RSD were less than 13%.7) A novel antibody coated competitive fluorescence immunoassay with sequential saturation for detection of dibutyl o-phthalate was described. Under-best conditions, DBP can be determined in the concentration range of 0.1～300μg/L with a detection limit of 0.02μg/L. The regression equation of this assay is△F=6206.5-744.7logC(μg/L) and coefficient of correlation R~2 is 0.997. Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique and the cross reactivity rates were less than 10%. The accuracy of the assay has been evaluated using tap water, water samples from Suzhou River (Shanghai, China), drinking water, water leaching from disposable plastic products and spiked samples, recovery rates were 90.7%～108.8%,and RSD were less than 13%.8) A novel antibody coated competitive fluorescence immunoassay with sequential saturation for detection of dicyclohexyl phthalate (DCHP) was described. Under best conditions, DCHP can be determined in the concentration range of 0.01～100μg/L with a detection limit of 0.01μg/L. The regression equation of this assay is△F=4127.2-550.2logC(μg/L) and coefficient of correlation R~2 is 0.998. Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique and the cross reactivity rates were less than 7%. The accuracy of the assay has been evaluated using tap water, water samples from Suzhou River (Shanghai, China), drinking water, water leaching from disposable plastic products and spiked samples, recovery rates were 90.6 %～109.5%,and RSD were less than 11%.9) A novel antibody coated competitive fluorescence immunoassay with the equilibrium method for detection of dicyclohexyl phthalate was described. Under best conditions, DCHP can be determined in the concentration range of 0.01～100μg/L with a detection limit of 0.01μg/L. The regression equation of this assay is△F=3846.1-422.3logC(μg/L) and coefficient of correlation R~2 is 0.997. Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique and the cross reactivity rates were less than 12%. The accuracy of the assay has been evaluated using tap water, water samples from Suzhou River (Shanghai, China), drinking water, water leaching from disposable plastic products and spiked samples, recovery rates were 92.3 %～108.4%,and RSD were less than 12%.
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