| In recent years, the issues of environmental endocrine disruptors (EEDs) have attracted more and more public attention. During the past years, vast quantities of diverse chemicals have entered the environment in order to increase agricultural productivity and modern industrial process. However, many chemicals (e.g. bisphenol A,2,4-Dichlorophenoxyacetic acid, chlorpyrifos) in the environment have weak hormonal activity. Their lipophilic nature and long half-life allow them to accumulate and persist in fatty tissues of body. These small organic molecules might affect the development of any organism and the later functioning in adult life of an individual, a population and even a community.Thus, it is urgent to develop highly sensitive, selective, large-scale and rapid analytical methods to routinely screen endocrine-disrupting chemicals level in the environment. Conventional analytical methods used to detect EEP, such as high-performance liquid chromatography, gas chromatography, and mass spectroscopy, require expensive equipment, time-consuming and labor-intensive that may be difficult to field-deploy. To date, many Immunochemical techniques have been established for analyzing environmental endocrine disruptors in miscellaneous samples duo to their fast, highly selective and cheap characteristics. However, most of these reports describe the detection of a single analyte.Liquid Immunoarray (LIA) is simply a transfer of the microarray format from a glass slide (planar and solid microarray) to a high-throughput and efficient microsphere format. Recent developments have given this technology the potential to be an inexpensive and flexible tool. The assays have been developed on a commercially available flow cytometer, the Luminex LX-100 (Luminex Corp, Austin, TX). It is an open platform and has been widely used in biomedical filed.Based on successful preparation for antibody against BPA and coating antigen, we have developed Luminex immunoassays that are rapid, sensitive, and specific and can detect 2,4-D, CHI and BPA simultaneously in a single test sample. Assay results obtained from our assays were compared to ELISA and HPLC.Three complete antigen for BVA,2,4-D and CHI were sucessfully produced by DCC method and the reaction products were characterized by UV-Vis, FT-IR and mass spectroscopy. The results obtained by mass spectrometry analysis show that the mole coupling ratio of BVA-BSA,2,4-D-BSA and CHI-BSA were 15:1,22:1 and 11:1 respectively. Based on Mannich reaction the complete antigen for BPA were sucessfully produced by conjugation between BPA with cationized bovine serum albumin (cBSA) and the reaction products were characterized by mass spectroscopy. The results obtained by mass spectrometry analysis show that the mole coupling ratio of BPA-cBSA was 14:1.BALB/c mices was immunized with two different immunogen. The result show that BPA-cBSA as immunogen could significantly enhance the immune response against BPA compared with BVA-BSA groups. We also observed that the similar sensitivity of antisera against BPA between BPA-cBSA group and BHPVA-BSA group.Based on antibody and coating protein (CP), ELISA standard curves for BPA,2,4-D and CHI were draw. The linear range were at 40-1289,34-1914 and 38-234 ng/mL respectively; The IC50 values (50%B/Bo) were 177,366 and 108 ng/mL respectively; the limit of detection (LOD) were approx.16,3 and 8ng/mL respectively.Recoveries of BPA (400,100 and 25 ng/mL),2,4-D (800,200 and 50 ng/mL) and CHI (200,100 and 50 ng/mL) from blank river water and tap water averaged between 68%-142%, 55%-135%and 90%-108%respectively; the average coefficients of variation (CV) ranged from 6%-15%,6%-14%and 3%-12%respectively.Based on conventional ELISA, we draw direct hapten conjugation ELISA (dhc-ELISA) standard curve (R2=0.993). The IC50 and the LOD for BPA were 23.5ng/mL and 0.3ng/mL respectively. Recoveries of BPA (250,50 and 10 ng/mL) from blank river water and tap water averaged between 60%-132%and the average CV ranged from 6.7%-13.6%.The results indicated that dhc-ELISA could improve assay sensitivity compared with conventional ELISA.After carefully optimizing, we draw Luminex standard curves for BPA,2,4-D and CHI. The linear range were 0.8-246.1,5.4-391.1 and 14.2-364.9 ng/mL respectively; the IC50 were approx.30.8,64.0 and 98.3 ng/mL respectively; the LOD were 0.03,0.35 and 0.16 ng/mL respectively. To investigate Luminex technology for simultaneous detection of EEDs in real sample, the 18 different samples were prepared by adding BPA (10,50 and 150 ng/mL),2,4-D (20,100 and 300 ng/mL) and CHI(30,150 and 450 ng/mL) to blank river water or tap water. When blank water samples contained one target compound, recoveries of BPA,2,4-D and CHI averaged between 41%-132%,64%-147%and 52%-109%respectively; the average CV ranged from 0.8%-8.7%,0.3%-9.6%and 1.7%-8.8%respectively. When blank water samples contained two target compounds, recoveries of BPA,2,4-D and CHI averaged between 52%-123%,51%-121%and 64%-97%respectively; the average CV ranged from 0.9%-9.9%,0.6%-6.0%and 0.8%-5.8%respectively. When blank water samples contained three target compounds, recoveries of BPA,2,4-D and CHI averaged between 43%-66%, 62%-112%and 51%-68%respectively; the average CV ranged from 3.2%-5.5%, 1.1%-9.3%and 2.2%-7.1%respectively.
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