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The development of an of endocrine disrupting compound detection system
Expanded Title:Certain hormone active agents in the environment can disrupt chemical messengers (hormones) of the endocrine system by sending erroneous signals or blocking legitimate signals. Also known as endocrine disrupting compounds (EDCs), these chemicals exert their deleterious effects on humans and wildlife by mimicking, blocking and disrupting the physiological functions of hormones. Hormones exert their functions by interacting with their corresponding receptors in target cells to trigger responses and prompt normal biological functions such as growth, development, behaviour and reproduction. Interference with the activities of hormones, such as with EDCs, can to stunted growth, impairment of short term memory, tubal pregnancy, low sperm count, reproductive failure and damage of the immune system. The hydrophobicity of EDCs and other chemical properties, has created unique challenges to developing techniques for detecting and screening these compounds. Detection methods include solid phase extraction (SPE) followed by: high performance liquid chromatography, liquid chromatography/mass spectrophotometry or gas chromatography/mass spectrophotometry. These techniques are, however, limited for general EDC monitoring due to high instrument costs, intensive labour and, in some instances, relatively poor sensitivity. In addition high-end analytical procedures are usually specific for one single analyte only, or a limited class of structurally related compounds. We proposed the development of a dip stick system based on the following principles. The ligand binding domain of the hERaLBD and the hARLBD are immobilised on a membrane contactor matrix using noncovalent AC technology. The immobilised receptor is exposed to the water containing low concentrations of estrogenic and/or androgenic compounds. These compounds are bound to the corresponding receptor and concentrated on the contactor surface through the receptor ligand interaction. After activation — a mild increase in temperature — the hERaLBD- or HARLBD estrogenic/androgenic compound-complex can be indicated using specific antibodies in an enzyme linked immuno-assay system. The presence of estrogenic/androgenic compounds will be indicated by the development of a specific colour on the contactor strips. The project involved membrane fabrication, receptor expression, receptor purification and antibody production against fusion receptors, and finally binding studies. Membranes were fabricated from polysulphone (PSU), polyether imide (PEI) and polyvinylidene fluoride (PVDF. An amylose functionalized cellulose acetate (CA) membrane was also fabricated. A Pluronic® F108 modified linker was synthesized. The receptor expression was achieved by sublconing the ligand binding domain of the steroid receptors into a suitable vector. The hERaLBD protein was expressed in E. coli BL21 (DE3) pLysS. The truncated human androgen receptor ligand binding domain, hARLBD, was subcloned into pTrcHis, a plasmid. The construct (AR-pTrcHis) was used to transform TOP10 E. coli. Expression and purification of the hARLBD were almost identical to that of the hERaLBD Purification of the hERaLBD protein showed efficient binding. The proteins were purified on an amylose column. The coupled fusion protein was eluted and the purified hERaLBD inoculated into a rabbit to generate polyclonal antibodies against the hERaLBD. For estrogen binding studies, PVDF membranes and polystyrene microtitre plates were used as a scaffold for the Pluronic-DMDDO. Wells of a plate were incubated with Pluronic-DMDDO and Pluronic F108. Purified hERaLBD and hARLBD solutions were then added and a solution of estradiol was pipetted into each well. After incubation, elution buffer was applied. Radioactivity of each sample was measured. The binding studies showed that significantly higher binding of radioactive estrogen could be shown in the wells coated with Pluronic-DMDDO charged with hERaLBD, indicating immobilization of hERaLBD. The Pluronic- DMDDO experiment charged with his6-ARLBD, yielded similar results. These experiments clearly indicate that not only was the ligand binding domains of the androgen and estrogen receptors immobilized, but the receptors retained bioactivity by binding to androgenic and estrogenic compounds in water solutions, respectively. The results conclusively demonstrate that the affinity immobilisation of estrogenic and androgenic compounds via the ligand binding domain of the human estrogen and androgen receptors on inert membrane contactors is possible and practically feasible.
Date Published:01/06/2013
Document Type:Research Report
Document Subjects:Drinking water - Water supply
Document Keywords:Water Quality
Document Format:Report
Document File Type:pdf
Research Report Type:Standard
WRC Report No:1534/1/13
ISBN No:978-1-4312-0074-0
Authors:Swart P; Govender S; Asongwe L; Truebody B; Jacobs EP
Project No:K5/1534
Organizations:University of Stellenbosch
Document Size:2 288 KB
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