Rapid and Sensitive Enzyme-Linked Immunosorbent Assays (ELISAs) for Selected Potent Endocrine Disrupting Steroids: Development and Application to Environmental Studies Chatchaporn Uraipong Thesis submitted in partial fulfillment of the requirement for the Degree of Master of Science (Research) School of Chemical Engineering The University of New South Wales September, 2010 ABSTRACT Endocrine 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 i 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 -1 -1 presented with a LOD and IC50 value at 0.07 ng L and 0.4 ng L , 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. ii ACKNOWLEDGEMENTS I would like to express my thanks to my supervisor Dr. Nanju Alice Lee for providing me with an opportunity to conduct research in the School of Chemical Engineering, Food Science and Technology. Her innovative research direction, valuable advice, and enthusiastic supervision throughout the course of this study are much appreciated. Sincere thanks also to Dr. Victor Wong for his timely help in writing skills as well as scientific advice. I would like to thank Mr. Camillo Taraborrelli and Mr. Eryanto Yanasuwan for their kind technical assistance on my work. Further thanks also to Dr. Chunhua Li of the University of Sydney, for her valuable water samples/extracts and Miss Christine Tan of the University of NSW for her valuable haptens. The ARC is especially acknowledged for funding of my research at the University of New South Wales (ARC Discovery DP0559179). The Glaxo Wellcome and Dr E. Routledge, for their kind gift of the recombinant yeasts to Dr. Alice Lee are also acknowledged. My sincere gratitude goes to my family for their ongoing encouragement and beliefs in me. I also would like to specially thank Jay and Molly for their unconditional love and prayer throughout my study. Thanks also to all of my friends. Their cooperative and friendly manner has created a stress- free working environment. Finally, to others who were not mentioned, your help and support are much appreciated, for without you, this thesis could not have been completed. iii LIST OF PUBLICATIONS IN PREPARATION C. Uraipong, C. H. Tan , V. Wong , C. Li, I. R. Kennedy, R. D. Allan & N. A. Lee, (2011) Sensitive and specific competitive enzyme immunoassays for monitoring of a potential endocrine disrupting hormone, 17α-ethynylestradiol and mestranol, in aquatic system (Journal of Food and Agricultural Chemistry) C. Uraipong, C. H. Tan , V. Wong , C. Li, I. R. Kennedy, R. D. Allan & N. A. Lee, (2011) Highly sensitive competitive enzyme immunoassays for monitoring of a potential endocrine disrupting synthetic hormone, mestranol, in aquatic system (Analytical and Bioanalytical Methods) C. Uraipong, C. H. Tan , V. Wong , C. Li, I. R. Kennedy, R. D. Allan & N. A. Lee (2011) Sensitive and specific competitive enzyme immunoassays for monitoring of a potential endocrine disrupting hormone, 17β-estradiol, in aquatic system (Analytica Chimica Acta) C. Uraipong, C. H. Tan , V. Wong & N. A. Lee (2011) Development of testosterone specific competitive enzyme immunoassay for monitoring in water quality (Analytical and Bioanalytical Methods) C. Uraipong, C. Li, I. R. Kennedy, Victor Wong & N. A. Lee (2011) Androgenic Activity in Water Bodies of selected urban and rural areas of New South Wales of Australia (Environmental Toxicology and Chemistry) iv LIST OF PRESENTATION N.A. Lee, C. Uraipong, C.H. Tan, V. Wong, C. Li, I.P. Kennedy, & R.D. Allan, Enzyme- Linked ImmunoSorbent Assay (ELISA) as a water Quality Monitoring Tool: Targeting a Potential Endocrine Disrupting Steroids, 17α-ethynylestradiol (EE2) IUPAC/RACI Conference (Chemistry for a Sustainable World, Melbourne 4th – 8th July 2010) N.A. Lee, C. Uraipong, C.H. Tan and V. Wong, Antibodies for characterizing and tracking of potential endocrine disrupting chemicals (ECDs) Pacifichem 2010 (International Chemical Congress of Pacific Basin Societies, Honolulu, Hawaii, USA 15th – 20th December 2010) v ABBREVIATIONS Ab purified antibody APCI atmospheric pressure chemical ionization BSA bovine serum albumin CBT checkerboard titration DCC N,N′-dicyclohexylcarbodiimide DDT dichlorodiphenyltrichloroethane DES diethylstilbestrol DMF dimethylformamide DMSO dimethyl sulfoxide E1 estrone E2 17β-estradiol E2-ACT polyclonal antibody of 17β-estradiol-acetate E2-ACT-BSA conjugate of 17β-estradiol-acetate and bovine serum E2-ACT-HRP conjugate of 17β-estradiol-acetate and horseradish peroxidase E2-ACT-KLH conjugate of 17β-estradiol-acetate keyhole limpet haemocyanin E2-BUT polyclonal antibody of 17β-estradiol-butyrate E2-BUT-BSA conjugate of 17β-estradiol-butyrate and bovine