Synthesis Of Artificial Antigen For Bisphenol A And Enzyme Linked Immunosorbent Assay Method

Bisphenol A(BPA)is able to mimic the effects of endogenous hormones and influences the central nervous system by combining with estrogen acceptor or effecting transmission approach of cell signal in human being as analogous chemical structure as estrogen.It shows irritation to skin, respiratory tract,digestive tract and cornea.Cumulation of bisphenol A in human body may causes chronic toxicosis,and shows symptoms such as dizziness,skin rash,spirit discomfort,diarrhea and so on.In recent years, BPA has largely arisen in our environment,especially been found that BPA would released from many kinds of polycarbonate plastics for food package, infant feeding-bottles and barrels for drinking water.BPA might be harmful to human health.Therefor,study on preparation of the simple and fast Gold-labeled immunochromatography test slip or ELISA test kit plays an great significant role for protecting customers' health and food safety testing.This study focus on synthesizing the artificial antigen for BPA and establishing Enzyme Linked Immunosorbent Assay.The main contents and results are as follows:Above all,the structure of BPA has been modified,carboxyl group was introduced,then the carboxyl was conjugated to the carrier protein BSA via amino bond by EDC and NHS method.UV and IR spectrum were used for identification of artificial antigen,The UV spectrum shows that characteristic absorbance bands of BPA and bovine serum albumin could be found on the UV spectrum of artificial antigen for BPA.The IR spectrum absorbance bands of bovine serum albumin and BPA were appeared on the IR spectrum graph of artificial antigen of BPA,moreover,a shrinked and vibration bands at 1641.2cm^-1and 1376.5cm^-1showed C=O and C-N bonds were produced and suggested the modified BPA and BSA had been linked. The results showed that the artificial antigen for BPA was synthesized successfully.Polyclonal antibody was obtained by immunizing Balb/C mice,and used in an indirect enzyme-linked immunosorbent assay for the determination of antibody titre.The final antibody titre is 256000,the optimum working concentration is 1:4000,the optimum coating concentration is 10μg·mL^-1, incubated at 37℃for 1h then coating at 4℃for 12h is better.Indirect competitive enzyme-linked immunosorbent assay determination results shows that the linear range of bisphenol A calibration curve was 10μg·L^-1～1×10⁵μg·L^-1,The regression equation of the curve is y=0.1038x+ 0.0007,correlation coefficient R²=0.9761,its detection range is 83.18～5.01×10⁷μg·L^-1,the result presents an inhibition concentration at 50% (IC50)of 64565μg·L^-1,and the limit of detection(LOD)is 9.934μg·L^-1. The specificity of antiserum was proved well because the across reactivities with benzene,tert-Butylbenzene,hydroquinone and o-hydroxybenzoic acid were found lower than 0.01%,except phenol was 0.06%.This determination method has already been applied in detection of bisphenol A in barreled drinking water,river water samples and surface water of factory,factual results shows BPA could release from the polycarbonate barrel,and concentration shows a rising trend as time increased,dissolved quantity detected after 30 days exceed 300μg·L^-1,the recoveries are 88.03～100.91%.BPA haven't been detected in river water samples and its recoveries are 98.46%～100.65%.Two surface water of factory which used BPA were determined that the concentration of BPA were 114μg·L^-1and 304μg·L^-1respectively,corresponding recoveries are 89.10～100.16%and 93.42～100.49%.The corresponding concentration of BPA detected by UV assay was almost approximate as the results showed by the ELASA determination.It indicated the results' consistency through both the two methods.The recoveries determination results show good veracity, and this method could be applied to qualitative and quantitive detection for bisphenol A in environmental water samples.