Study On The Immunoassays Of TBBP-DHEE And TBBPA-MHEE And Their Applications In Risk Assessment

Tetrabromobisphenol A flame retardant（TBBPA）is a kind of Brominated flame retardant（BFRs）,which is widely used in electronic products,paints and textiles in the daily life as additive flame retardants.TBBPAs enters the environmental medium through various channels and influences the organism in the environment.TBBPAs have shown endocrine disrupting toxicity,neurotoxicity and so on,which has potential risks to environmental ecology and human health.Tetrabromobisphenol A bis（2-hydroxyethyl）ether（TBBPA-DHEE）isanimportantderivativeof Tetrabromobisphenol A and tetrabromobisphenol A mono（hydroxyethyl）ether（TBBPA-MHEE）is a byproduct of TBBPA-DHEE.Conventional instruments analysis methods need complex pretreatment,expensive equipment and professional operator,which is not suitable for large samples.Therefore,it is urgent to establish a high throughput,low cost and short time method to provide technical support for detecting and analyzing the concentration and distubution of TBBPA compounds in environment.This study designed and synthesized hapten of TBBPA-DHEE,developed and screened monoclonal antibodies,established three ELISA methods for recognizing two compounds specifically or simultaneously,investigated the pollution of two compounds and assessed the risk of two compounds in regional.The main research concents were as follows:（1）The hapten（D₃）was prepared and conjugated with carrier protein for coating antigen（D₃-OVA）and immunogen（D₃-BSA）.After immunied Balb/C mice with D₃-BSA,the spleen cells of immunized mouse with highest sensitivity were fused with SP2/0 murine mycloma cells.After screening and cloning,five hybridoma cell lines that steadily produced monoclonal antibody（mAb）against TBBPA-DHEE were selected:D2D2,D2D4,D2D4,D4G6,F7D5.The immunoglobulin subtypes of the five monoclonal antibodies were identified as IgG1 with Kappa light chain.（2）On the basis of the previous research,analysing the antigen-antibody pairs of five hybridoma cell lines with D₃-OVA,and M₃-OVA.WhenD₃-OVA was used as coating antigen,it was found that five mAb were able to recognize TBBPA-DHEE and TBBPA-MHEE.When M₃-OVA was used as coating antigen,mAb-D4G6 could recognize TBBPA-DHEE specifically,and mAb-D2D6 could recognize TBBP-MHEE specifically.（3）Based on the result of antigen-antibody,three modes were established:Format1,M₃-OVA/mAb-D4G6;Format2,M₃-OVA/mAb-D2D6;Format three:D₃-OVA/mAb-D4G6.The condition parameters of threewere optimized and the standard curves were established.For Format 1,the detection limit（LOD）and IC₅₀0 were 0.12 ng/mL and 2.53 ng/mL,the liner range was 0.51-12.56 ng/mL.For format 2,LOD)and IC₅₀0 were 0.79 ng/mL and 6.34 ng/mL,the liner range was1.31-30.76 ng/mL.For formate 3,LOD were 0.53 ng/mL and 0.68 ng/mL,respectively,IC50 were 2.56 ng/mL and 3.11 ng/mL,respectively,liner range were0.85-7.7 ng/mL and 0.76-12.7 ng/mL respectively.（4）The samples in typical areas were collected,including the e-waste dismantling area（Taizhou city,zhejiang province）,manufacturers of BFRs（Shouguang city,shandong province and Liuyang city,hunan province）.The three ELISA methods weie applied to detect the concentration of two compounds in environmental samples.The detection rate of TBBPA-DHEE in water samples soil samples from Taizhou city was 73.91%and 83.33%.The detection rate of TBBPA-MHEE is low,taizhou city is polluted by TBBPA-DHEE.The detection rate of samples from Shouguang city was100%and the highest concentration is 4.02ng/mL,which was higher than that form Taizhou city.TBBPA-DHEE and TBBPA-MHEE wree not detected in water samples from Liuyang,only TBBPA-DHEE was detected in samples near the effluent outlet of the factory.The potential risks of two compounds in three areas were assessed by ambient severity（AS）and health risk assessment（HSR）.It is impossible to assess the potential risk of two compounds by AS.It was found that the health index of children is higher than adult by combining AS with HSR.Therefore,children are impacted by TBBPA-DHEE and TBBPA-MHEE more easily.