The Research On Preparation And Application Of Antibodies Of Persistent Organic Pollutants

Persistent organic pollutants(POPs) are a kind of synthetic chemicals, which are highly toxic, semivolatile, persistent and bioaccumulative. They can migrate long distance through migratory species in the atmosphere and water, then deposit back to the earth. POPs are accumulated through biological food chain and pose harmful effect to human health and the environment.At present, analysis methods of POPs are mainly based on gas/liquid chromatography(GC/LC), high-performance liquid chromatography(HPLC)-tandem mass spectrometry(MS), as well as GC-MS. Although these methods are suitable for analyzing complex materials and have high accuracy, they require expensive instruments, extensive sample pretreatment and are not conducive to on-site rapid analysis of large quantities of samples. So developing an accurate, fast and sensitive detection method of POPs in daily necessities, cosmetics, food and the environment is extremely urgent. In recent years, immunoassay has been widely applied for environmental monitoring field. At the same time, the growing emergence of new methods and new means have laid the solid foundation for developing immunoassay of POPs. The immunoassay method is low cost, simple, fast, sensitive and suitable for analyzing trace components of complex matrices. However, POPs are small toxic molecules and it is hard to develop their antibodies. At present, the quantities of POPs which can be analyed by immunoassay are small. There are no reports about the immunoassay of2,3’,4,5’,6-pentabromodiphenylether(BDE-121) and tris(2,3-dibromopropyl)ocyanurate(TBC).Based on the situation above, this thesis aimed to establish rapid analysis method of POPs, choosed BDE-121and TBC as the research objects, developed the studies of their related immune analysis methods. The main research work are as follows:(1) Preparation of anti-BDE-121polyclonal antibody and their applicationThe artificial antigen (BDE-121-BSA) was prepared by using the BDE-121derivative which was designed and synthezied by our research group. Through immunising rabbits, the anti-BDE-121polyclonal antibody was developed. The indirect competitive enzyme-linked immunoadsorption assay method (IC-ELISA) was established with the anti-BDE-121polyclonal antibody. The experimental conditions including the concentration of the coating antigen and antiserum, pH, ionic strength, the type and concentration of organic solvent of the buffer solution were optimized. The optimized ELISA conditions are as follows:coating antigen concentration of10μg/mL, antiserum at a dilution of1:8000in wells, ionic strength of0.04M and20%methanol in PBST (pH7.4) assay buffer. Under these optimized conditions, a linear range (IC20-80) of1.74to84.1μg/L of this method was obtained and the IC50value was8.07μg/L. The limit of detection (LOD, IC10) was defined as0.644μg/L. Twelve kinds of compounds were tested for calculating cross-reactivities (CR%), all of the tested compounds show little CRs with BDE-121(<9%). These results indicated that the specificity of the developed antibody was high. The furniture foam, sera (human) and paint sample spiked with BDE-121were analyzed by the proposed ELISA and GC/MS, respectively. The results were basically identical and meet the experiment requirements.(2) Preparation of anti-BDE-121monoclonal antibody and the signal amplification method for detection of BDE-121The artificial antigen (BDE-121-OVA) was prepared by using the BDE-121derivative which was designed and synthezied by our research group. Through immunising mice, the anti-BDE-121monoclonal antibody was developed. The experimental conditions were optimized with the monoclonal antibody and the IC-ELISA in the second chapter. The optimized conditions are as follows:coating antigen concentration of10μg/mL, antiserum at a dilution of1:10000in wells, ionic strength of0.03M and15%methanol in PBST (pH7.4) assay buffer. Under these optimized conditions, the HRP goat anti-mouse IgG and the HRP goat anti-mouse IgG which modified with gold nanoparticles were used to detect BDE-121, respectively. The IC50values of the two methods were3.52μg/L and2.78μg/L and the sensitivity was improved by21.02%. Twelve kinds of compounds were tested for calculating cross-reactivities(CR%), all of the tested compounds show little CRs with BDE-121(<3.6%). These results indicated that the specificity of the developed antibody was higher than the polyclonal antibody. The furniture foam, sera (human), and paint sample spiked with BDE-121were analyzed. The recoveries were between93%-110%. These results indicated that the developed monoclonal antibody can be used for the detection of BDE-121in the environment.(3) Preparation of anti-TBC monoclonal antibody and their applicationThe artificial antigens (TBC-Tcl-OVA, TBC-Tc2-OVA, TBC-Tc3-OVA) were prepared by using the TBC derivatives which were designed and synthezied by our research group. Through immunising mice, the anti-TBC monoclonal antibody was developed. The experimental conditions were optimized with the monoclonal antibody and the IC-ELISA in the second chapter. The optimized conditions are as follows:coating antigen concentration of10μg/mL, antibody at a concentration of50ng/mL in wells, ionic strength of0.2M and25%methanol in PBST (pH7.4) assay buffer. Under the optimized conditions, the IC50value was1.59μg/L and the limit of detection (LOD) was defined as0.06μg/L. Sixteen kinds of compounds were tested for calculating cross-reactivities (CR%), all of the tested compounds show little CRs with TBC (<2%). The CRs were lower compared with the polyclonal antibody which prepared by our group before. The tap water, waster water, soil samples and sera (human) spiked with TBC were analyzed by the proposed ELISA and UHPLC-MS/MS. The results indicated that the method is valid and can be used for the detection of TBC in the environment.