Study On The Immunoassays Of TBBPA-DAE And Its Primary Application In Risk Assessment

Tetrabromobisphenol A（TBBPA）is one of the typical brominated flame retardants（BFRs）,which has been widely used in textiles,electronic products,paints and other products.TBBPA enters into the environmental through various ways and has an impact on the living body in the environment.It was found that TBBPA compounds presented chronic toxicity such as cytotoxicity、neurotoxicity and had risks to environmental ecology and human health.Tetrabromobisphenol A diallyl ether（TBBPA-DAE）is an important derivative of TBBPA.Few studies have been reported to explore its occurrence in environment and evaluate its ecological risk.Analysis of these compounds often based on instrument method.However,these methods need expensive instrument and professional operators and the sample preparation procedures are tedious,which hindered the use of chromatography methods in screening pollutants in environmental samples.Therefore,it is urgent to establish convenient low-cost and time-consuming analysis methods for TBBPA-DAE.In this study,using TBBPA-DAE as target four hapten were designed and synthesized,monoclonal antibody against TBBPA-DAE was screened and prepared,ELISA was established to detect TBBPA-DAE specifically;the ecological risks were evaluated according to the results from water samples from typical regions;further,new immunochromatographys were developed based on PAMAM-Au for rapid screening of targets.The specific research contents were as follows:（1）TBBPA-DAE haptens（D₂,D₆,M₂,M₆）were prepared and identified.The haptens were coupled with carrier protein to prepare immunogen and coated antigen.Balb/C mice were immunized with immunogen and the spleen cells of mice were fused with SP2/0.Three cell lines with stable secretion of monoclonal antibody TBBPA-DAE were prepared and evaluated accordingly.（2）The model of ELISA was established using M₆-OVA as coating antigen and E7C2 as specific antibody.After optimization of reaction condition,including coating time,response time,the standard curve was established.（3）The water samples of specificial pollution areas were collected,including Taizhou City e-waste dismantling site,Shouguang City and Liuyang City of BFR manufacturer.The concentration of TBBPA-DAE in regional environmental samples was detected by established by ELISA analysis method.（4）An immunochromatographic method based on PAMAM-Au was established.The results of semi-quantitative analysis were quantitatively analyzed.Based on the above research,the main conclusions are as follows:（1）Three cell lines E7C2,D5E7G3,4E3,which stably secreted TBBPA-DAE monoclonal antibody were screened and cloned.The results showed that the heavy chains of the monoclonal antibodies were IgG1 and the light chains were Kappa.The cross-reaction rate of E7C2 cell line to TBBPA and other derivatives were less than0.01%.（2）The ELISA established in this study with IC₅₀0 value was 0.17μg/mL,LOD was0.031μg/mL,the linear range was 0.078⁰.41μg/mL,and correlation coefficient R²≥0.99.According to the recovery experiment,the recovery rates of TBBPA-DAE was97.5¹07.5%.The established method was used to detect the samples.In 15 water samples from Taizhou,the detection rate of TBBPA-DAE is 40%,and the detected concentration range is ND⁰.0481μg/mL.TBBPA-DAE was detected in only one of the five samples collected in Shandong Province.TBBPA-DAE compounds were not detected in water samples collected near Liuyang River,Hunan Province.The data of assessment showed that TBBPA-DAE has little impact on health and ecological environment,and has low potential risk factors.（3）The IC₅₀0 and LOD of immunochromatography based on PAMAM-Au sensor were 0.192μg/mL and 0.048μg/mL,respectively.Further,enhanced immunochromatographic method based on PAMAM-Au-HRP was developed,which IC₅₀0 and LOD were 7 ng/mL and 1.5 ng/mL,respectively.The sensitivity of immunochromatography based on PAMAM-Au-HRP was increased about 25 times.In this study,monoclonal antibodies of TBBPA-DAE were successfully prepared,and the method of rapid detection of TBBPA-DAE was established;The synthetic particles were prepared which applied to the immunochromatographic strips to realize the rapid visual detection of pollutants in the environment.