Production Of Antibody Against Two Tetrabromobisphenol A Analogues And Development Of Immunoassay

Tetrabromobisphenol A(TBBPA) is a new brominated flame retardant, which was synthesized from bisphenol A. Because of their outstanding properties in flame retardance, high thermal stability and low cost so it was widely used in the industrial production and electronic equipment. However, some research showed that TBBPA could cause side effects to human and wildlifes, and it also was regarded as a persistant chemical with hgih bioaccumulation and trophic transfer. So it is necessary to develop an efficient and reliable method to investigate the distribution, transportation and risk assessment in environments.This research studied the synthesis of the hapten of TBBPA and Tetrabromobisphenol A 2-hydroxyethyl ether(TBBPA-DHEE), and then produced the corresponding polyclonal antibodies based on which, novel rapid and high- throughput ELISA methods were established to detect TBBPA and TBBPA-DHEE, respectively. And some parameters that could influence the performance of the method also were investigated. At the same time, considering that Tetrabromobisphenol A diallyl ether(TBBPA-DAE) and tetrabromobisphenol A bis(2,3-dibromopropyl ether)(TBBPA-DBPE) can not be purchased, we synthesized the chemicals, which is needed in cross-reactivity study. And the main results of our studies were outlined as following:(1) Hapten TBBPA-D1 was obtained by steps of esterification, bromination and hydrolysis using diphenolic acid as raw material. Hapten TBBPA-D2 was generated through addition reaction, esterification, bromination and hydrolysis with bisphenol-A and 5-acetyl pentanoic acid as raw materials. And then immunogen(TBBPA-D2-BSA) and coating antigen(TBBPA-D2-OVA) was obtained by coupling macromolecular carrier protein(BSA and OVA), respectively.(2) Based on the haptens, immunogens(TBBPA-D1-BSA, TBBPA-D2-BSA) and coating antigen(TBBPA-D1-OVA, TBBPA-D2-OVA) were produced by coupling carrier proteins(BSA and OVA), respectively.(3) Hapten TBBPA-DHEE-M1 was synthesized by substitution reaction using TBBPA and bromoacetic acid as raw materials. Similary, immunogen(DHEE-M1-BSA) and coating antigen(DHEE-M1-OVA) were obtained by substitution reaction and binding carrier proteins(BSA and OVA), respectively.(4) TBBPA-DAE and TBBPA-DBPE was obtained by elimiaction using TBBPA as raw material. And then, authenticated the products by H-Nuclear-Magnetic-Resonance(HNMR).(5) TBBPA-D3 and TBBPA-D4 was obtained by addition reaction using TBBPA、bromoacetic acid and 3-bromopropionic acid as raw material. And then, authenticated the products by H-Nuclear-Magnetic-Resonance(HNMR).(6) After immuned New-zealand rabbits with immunogens(TBBPA-D2-BSA, TBBPA-D1-BSA), the polyclonal antibodies of R1-1 and R2-1 were generated. The antibodies showed higher specificity without cross-reactivity to any structre related chemicals. The detection limit of ELISA using R1-1 was 18.53 ng/mL with IC50 of 51.0 ng/mL.(7) Under the similar procedures as(2), a polyclonal antibody named R3-2 was produced, which could recognize simultaneously TBBPA-DHEE and TBBPA-MHEE. Using the antibody, an ELISA method was developed with the detection limit of 8.74 ng/m L and IC50 of 36.4 ng/mL.