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(2010)ThesisEndocrine disrupting chemicals (EDCs) are chemicals that alter functions of the endocrine system and cause health effects in an intact organism, or progeny, or population, with reproductive, developmental, or carcinogenic consequences. In order to facilitate risk assessment of potential endocrine disrupting steroids that are present in ultra low concentrations in the Australian environment, there is a need to boost the analytical capacity for EDC detection. One strategy is to develop antibody-based techniques that can offer simple, cost-effective and reliable analysis with high throughput capacity and portability for real-time monitoring. This thesis describes the design and synthesis of hapten molecules, raising of specific antibodies, formatting and characterising of a series of sensitive competitive Enzyme-Linked Immunosorbent Assays (ELISAs) for 17β-estradiol (E2), 17α-ethynylestradiol (EE2), ethylestradiol-3-methyl ether (mestranol) and testosterone (T), including validation of their performance as fast and effective water monitoring tools. Application of the developed assays to investigate the levels of the target EDCs in bodies of water and efficiency of water treatment plants in urban and rural areas in New South Wales, Australia, is also discussed. 17α-Ethynylestradiol and related synthetic estrogens, are active ingredients of contraceptive pills and hormone therapy, and have been identified as potent EDCs (Warner and Jenkins, 2007). In Chapter 3, the development of two ELISAs with varying specificity for the synthetic estrogens is described; 1) a highly sensitive ELISA for 17α-ethynylestradiol and mestranol, and 2) a mestranol specific ELISA. Highly specific antibodies was facilitated by synthesising haptens with the attachment of linkers with varying lengths at the C3 position (17α-ethynylestradiol-acetate hapten (EE2-ACT-KLH) and 17α-ethynylestradiol-butyrate hapten (EE2-BUT-KLH)). The optimised ELISAs in heterologous systems displayed high sensitivity, with the best assay exhibiting a limit of detection (LOD) of 70 ng L-1 EE2 in water (without preconcentration). The LOD of the ELISA covering the preconcentration step was 0.13 ng L-1 EE2 in water samples. This ELISA also correlated well with GC-MS (R2 = 0.934) data that were conducted independently for the same spiked samples. In Chapter 4, the development and validation of competitive direct ELISAs (cd ELISA) for the detection of 17β-estradiol (E2) in water at sub-parts per trillion levels is described. The specific polyclonal antibodies were raised against KLH conjugates of 17β-estradiol-acetate hapten (E2-ACT-KLH) and 17β-estradiol-butyrate hapten (E2-BUT-KLH), using a similar hapten synthesis approach as for 17α-ethynylestradiol. The developed ELISA (denoted as E2 ELISA) was highly specific to 17β-estradiol with a LOD of 50 ngL-1 in water (without preconcentration), with high matrix tolerance. The LOD of the E2 ELISA covering the preconcentration step was 0.03 ngL-1in water samples. Validation of ELISA performance against an independently performed GC-MS method indicated good correlation with a R2 value of 0.936. In Chapter 5, the development of an ELISA specific for testosterone with respect to the desired sensitivity and specificity for environmental analysis is described. The ELISA presented with a LOD and IC50 value at 0.07 ng L-1 and 0.4 ng L-1, respectively, and an excellent specificity. Unlike previously described assays for the estrogens, this assay was more matrix sensitive. Hence, an alternative or enhanced sample preparation step prior to assay was deemed essential to improve assay performance. Finally, in Chapter 6, the application of these developed ELISAs for the investigation of the quality of influent and effluents from various Sewage Treatment Plants (STPs) and nearby water resources in New South Wales, Australia, is described. The estrogenic and androgenic activities of the respective water samples were 0.20 to 6.28 ng L-1 estradiol equivalents and 0.01 to 0.64 ngL-1 testosterone equivalents, respectively, as measured by the yeast screening assays. Regular monitoring of EDCs should be conducted to ensure the levels and the contribution of estrogenic potency in water bodies is maintained below the ecotoxicologically significant level of 10 ngL-1 (Fenske et al., 2005, Huschek and Hansen, 2005). In addition, agricultural practices including live stock husbandry should be controlled to a certain distance away from water sources as a precautionary measure to reduce estrogenic potency in our water resources. In conclusion, a satisfactory agreement in data for E2, EE2 and T was obtained between ELISAs, GC-MS, and the estrogenic/androgenic activities measured by the yeast screening assays, although a slightly higher estimation was observed by ELISA. This suggests that ELISAs developed in this project can be used as fast and cost effective water quality monitoring tools. The high sensitivity and specificity of these ELISAs allow them to be used to monitor with acceptable reliability of the estrogenic and androgenic steroids at low parts per trillion levels after a simple concentration step.