| Dibutyl phthalate (DBP) is a kind of phthalate esters (PAEs), which is now widely used as plasticizers in industrial products, which include plastics, adhesives and lubricants. DBP is connected to other compounds through noncovalent bonds; it can be released to the environment very easily due to its low stability, and do harmful to the environment and human health. There are several ways for DBP to enter the human body, such as inhalation, skin contact and ingestion, sometimes medicial injection can be a high rsik exposure way. DBP can be accumulated in human body for a long time with a homeostasis way between intake and discharge. Research shows that DBP is an environmental endocrine disruptor, with reproductive toxicity, developmental toxicity and potential carcinogenic effects. In recent years, many technical methods can be used for measuring DBP, but the main methods are still the gas chromatography coupled with mass spectroscopy (GC-MS) and high performance liquid chromatography (HPLC), and these two methods are very difficult to operate, high testing cost and time consuming, so some better methods to estimate intake of phthalates, determining distribution and accumulation of DBP in biological materials are critically need. This research contains three parts.The first part is the development of ELISA test assay for detecting DBP. We firstly revived the hybridoma-cell-line which secretes monoclonal antibody aim at DBP, after three times’ screening by limiting dilution assays, a hybridoma cell line whit high sensitivity and high titer was got. In vivo induced ascites method and octylic acid ammonium sulfate methods were used to prepared and purified monoclonal antibodies of DBP. Then indirect competitive ELISA was optimized, such as the confining liquid, the concentration of BSA, reaction time and temperature, coloration system. Under the optimal conditions, an indirect competitive standard curve was established: y=15.401Ln(x)-24.13, R2=0.9973. It showed good relationship between the concentration of DBP and inhibition rate.In the second part, we applied the ELISA assay with a monoclonal antibody specific to DBP into biosamples’ tests. In our study, rats were exposed to DBP through oral route and dermal route for one week, and the concentration of DBP is 200mg/kg/d. The rats were killed after 24h,48h and 72h, getting the liver, kidney, blood and urine for the determination of DBP. The results show that, the content of DBP in rats exposed by oral route is higher than exposed by dermal route, there are significantly differences between these two routes and experimental groups vs control group. The content of DBP can be excreted by the body with urines gradually, after 72 hours, there is a little DBP left in organs.In the third part, we developed a novel method with a monoclonal antibody specific to DBP to determine the distribution and accumulation of tissue DBP in vivo. The contents of DBP in liver, kidney, stomach and testes were detected by immunofluorescence assays. These data give directly evidence that indicates the distribution and accumulation of DBP in vivo. Double-label immuno fluorescence assay provided with a visual approach to determination of the distribution and accumulation of DBP. It indicated that DBP accumulated in subcutaneous tissue such as sweat gland, hair follicle.Both of immunofluorescence assay and ELISA can be used to measure the content of DBP in biological materials. The developed assays showed that DBP accumulated in viscera being rich in fat, such as liver, kidney and could overcome physiological barriers to penetrate testes. The date suggested that the accumulations of DBP exposed through dermal route were less than that of oral route and most of DBP was metabolized in 2 or 3 days. |
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