| During recent decades much attention has been concentrated in the study of the health effects and risk assessment of many natural and synthetic substances in wildlife and humans. There are a large number of synthetic compounds known as environmental endocrine disruptors (EEDs) or environmental hormone that can interact in different ways with the endocrine system. It may affect the balance of normal hormonal function in organism. Nowadays environmental contamination by EEDs is a recognized worldwide problem.EEDs residue is one of the important monitoring items of safety in our environment. Analytical tools based on conventional techniques such as GC and LC coupled to sensitive detection systems such as MS have been applied to detect EEDs. However, traditional chromatogram monitoring methods is not fit for rapid and available inspection. Environmental monitoring generally requires to analyze a large number of samples, therefore, the development of a fast, sensitive and low cost analytical method for EEDs is primary interest. In the last few years, one of the most developed field testing methods has been immunochemical technique, it is one of the most competitive and challengeable monitoring methods because of its quickness, sensitiveness, economy and simple samples treatment.The purpose of our work is to develop new immunoassay methods for detection environmental hormones. For study of immunochemistry characteristic of EEDs, 2,4-dichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol were chosen as study objects. Haptens were synthesized and conjugated to preotein for rabbit immunization. Three kind of polyclonal antibodies were obtained. According as peculiarity of specific reaction between antigen and antibody, immunochemical method was established for detection chlorophenols residues in environmental samples. The method was showed rapid, sensitive, precise, and obtained satisfactory results as follows:1,2,4-Dichlorophenol hapten was synthesized by means of the diazo reaction, and 2,4,6-trichlorophenol or pentachlorophenol haptens were synthesized by means of the chloroactic acid method. The haptens were conjugated to the carrier proteins (BSA or OVA) with the modified active ester method or with the mixed acidanhydride method to form immune antigens and coating antigens. The conjugates were scanned by UV spectrum and SDS-PAGE to judge whether haptens were linked to the proteins, the results showed that the conjugates were prepared successful. The conjugation ratios of artificial antigens were within the range from 7:1 to 29:1.2,New Zealand white rabbits were immunized with the mixture of chlorophenols immunogen and Freund adjuvant, polyclonal antibodies of three chlorophneols were prepared successful. The titers of anti-sera were identification by tube agglutination reaction test, agar double-diffusion experiment and indirect ELISA method. The results showed that: in tube agglutination reaction test, there were visible precipitation reaction; in agar double-diffusion experiment, there were clear deposit lines; in ELISA experiment the titer of DCP anti-sera range from 3.2×10~4～1.28×10~5; TCP anti-sera range from 1.6×10~4～1.28×10~5 and PCP anti-sera range from 6.4×10~4～1.28×10~5. The immune sera were purified by octanoic acid-saturated ammonium sulfate, Sephadex G-50 and DEAE cellulose. Purified anti-sera were freeze-dried, aliquotted and stored at-20℃.3,By using the antibodies or label antibodies with high titer, three types of immunoassay methods have been established for 2,4-dichlorophenol detection, indirect ELISA, direct FIA and indirect FIA, respectively. The three calibration curve ranges were kept from 2 to 100μg/L, from 1 to 100μg/L and from 1 to 100μg/L with the three methods. The three linear regression curves in terms of concentration were as follows: I=20.21logC+24.13 (r=0.9917), F=21.57logC+26.83 (r=0.9898) and F =22.22logC+28.59 (r=0.9926). The limit of detection was 0.63μg/L, 0.41μg/L and 0.48μg/L, respectively. Compare with these methods, FIA method showed a wider calibration range and a lower sensitivity than ELISA method. The FIA methods were highly specific for 2,4-dichlorophenol and showed low cross-reactivity (CR) for other structurally related phenolic compounds (CR%＜5%). It showed a good accuracy and suitability to analyze 2,4-dichlorophenol in specimens of water and soil. By indirect FIA method, the recoveries of 2,4-dichlorophenol in tap water, river water and soil samples were range from 86.6 to 119.5%. The coefficient of variation was range from 2.87 to 6.53%. By direct FIA method, the recoveries of 2,4-dichlorophenol in tap water, river water and soil samples were range from 84.6 to 114.7%. The coefficient of variation was range from 1.73 to 7.46%.4,Conjugate of antibody and fluoresceine isothiocyanate (FITC) was prepared by direct label method for 2,4,6-trichlorophenol. According to the agar diffusion test, the titer of fluorescein labeled antibody reached 1:16. The direct FIA titer was 6.4×10~4. The F/P ratio of labeling reaction was 3.4. It showed that the label procedure was feasible. The labeled antibody was synthesized successful, and it was applicable to fluoroimmunoassay.By using the labeled antibody, two types of FIA methods have been established for 2,4,6-trichlorophenol detection, direct FIA and indirect FIA, respectively. The two calibration curve ranges were kept from 1 to 100μg/L and from 0.5 to 100μg/L for the two methods. The two linear regression curves in terms of concentration were as follows: F=27.16logC+20.42 (r=0.9938) and F=21.91logC+36.32 (r=0.9936). The limit of detection was 1.02μg/L and 0.18μg/L, respectively. The FIA method was highly specific for 2,4,6-trichlorophenol and showed low cross-reactivity for other structurally related phenolic compounds (CR%＜4%). It showed a good accuracy and suitability to analyze 2,4,6-trichlorophenol in specimens of water, soil and textile. By indirect FIA method, the recoveries of 2,4,6-trichlorophenol in water, soil and textile samples were range from 85 to 112.5%. The coefficient of variation was range from 1.92 to 9.54%. By direct FIA method, the recoveries of 2,4,6-trichlorophenol in water, soil and textile samples were range from 84.5 to 119%. The coefficient of variation was range from 2.05 to 7.94%.5,By using the antibody with high titer and optimized condition for reaction, indirect FIA has been established for pentachlorophenol detection. The calibration curve range was kept from 0.5 to 50μg/L. The linear regression curve in terms of concentration was F=4.15logC+33.03. The limit of detection was 0.29μg/L. The indirect FIA method is highly specific for pentachlorophenol and shows low cross-reactivity for other structurally related phenolic compounds (CR%＜6%).By the established indirect FIA, a FIA kit has been produced to detect the residue of pentachlorophenol in specimens of soil, wood and textile. Compare with the indirect FIA and GC methods, the FIA method showed the recoveries were ranged from 84.6 to 103.7%, with coefficient of variations of 4.18～8.28%, and GC method showed the average recoveries were ranged from 96.9 to 97.4%, with average coefficient of variations of 3.81～7.26%. It showed the indirect FIA method is very useful for monitoring pentachlorophenol compounds in environmental samples. The validity of kit was six months at least.
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