The Establishment And Study Of A Series Of Immunological Detection Methods For Bisphenol A

This paper aimed at detecting BPA in complex foods.Three methods of immunoaffinity column-high performance liquid chromatography?IAC-HPLC?,chemiluminescence enzyme linked immunosorbent assay?CLEIA?,immunoaffinity column-isotope dilution mass spectrometry?IAC-ID-UPLC-MS/MS?were established and evaluated.In addition,the preparation of anti bisphenol A?BPA?-anti acridine ester?AE?bispecific antibody is accomplished by chemical coupling method and cell fusion technique to develop a more convenient and effective immunochemistry luminescence method for BPAdetection.1.The establishment and evaluation of immunoaffinity column-high performance liquid chromatographyThe high performance liquid chromatography?HPLC?for detection BPA after purified with immunoaffinity column?IAC?was established.BPA was separated by Agilent C18 reversed phase column.The mobile phase for HPLC was methanol-water?55:45?.And the excitation and emission wavelengths were 227 nm and 313 nm,respectively,for FLD.In that conditions,the method showed good linearity with the BPA concentration ranging in 1-200 ng/ml.The average recoveries of the beer and milk samples range from 74.56%to 109.67%with the coefficient of variation range from 1.04%to 9.77%.The limit of detection?LOD,signal-to-noise,S/N=3?and the limit of quantification?LOQ,signal-to-noise,S/N=10?of beer samples were 0.28ng/ml and 0.95 ng/ml respectively.The limit of detection and the limit of quantification of milk samples were 0.5 ng/ml and 1.5ng/ml.2.Establishment and evaluation of chemiluminescent enzyme linked immunosorbent assay?CLEIA?The CLEIA was established to detect BPA.The effects of concentration of enzyme labeled anti-antibody,pH,ionic strength and concentration of organic solvent on the luminescent intensity of enzyme immunoassay?RLU?and half inhibitory concentration(IC₅₀)were studied.The optimal reaction conditions of antigen and antibody were chose according to the value RLU/IC₅₀.Through the optimization,the optimum reaction conditions of BPA antigen and anti-BPA antibody were that the concentration of coating antigen was 1ug/ml,enzyme labeled anti-antibody dilution ratio is 1:3000,dilution of BPA antibody with pH 8.0 0.01M PBS containing 0.8mol/L NaCl,standard solutions were prepared with ultra pure water.In the conditions,a competitive inhibition curve for?CLEIA?was established.The linear range is0.25-50 ng/ml,and the IC₅₀0 is 3.33 ng/ml.Recovery experiments were performed on three levels of milk samples,and the average recoveries range from 94.98%to112.91%with the coefficient of variation in the range of 0.93%-3.96%.3.Establishment and evaluation of immunoaffinity column-isotope dilution mass spectrometryAn ultra performance liquid chromatography-mass spectrometry?UPLC-MS?with an isotope bisphenol A-3,3',5,5'-d4?BPA-d4?used as internal standard after that the samples were purified with immunoaffinity column?IAC?was established for detection of BPA in beer,milk and canned fish.BPA was separated by Waters ACQUITY UPLCTM BEH C18.The mobile phase for UPLC-MS were methanol and0.1%ammonia with gradient elution.The ionization mode was electrospray ionization?ESI?and the data collected by multiple reaction monitoring negative ion mode.In that condition,the method showed good linearity with the BPA concentration ranging in 1-200 ng/ml.The average recoveries of the beer and milk samples range from 76.70%-112.91%with the coefficient of variation range from0.93%-14.34%.The LOD and the LOQ of beer samples were 0.33 ng/ml and 1 ng/ml respectively.The LOD and the LOQ of milk samples were 0.5 ng/ml and 1.5 ng/ml.The LOD and the LOQ of canned fish samples were 1 ng/ml and 3.5 ng/ml respectively.4.Preparation of anti BPA-aiti-acridine ester?AE?bispecific antibody by chemical conjunction.Coupling of anti BPA monoclonal antibody and anti AE monoclonal antibody by using bifunctional reagent sulfo-SMCC.The molar ratio of reactants,reaction time,and pH were optimized of the reaction.Results showed that the influence factors of the two reactions were molar ratio>pH>reaction time.According to the results of chemiluminescence and SDS-page,the anti BPA-anti AE bispecific antibody was prepared successfully.5.Preparation of anti BPA-acridine ester?AE?bispecific antibody by cell fusion techniquePrepare anti BPA-anti acridine ester?AE?bispecific antibody by three hybridoma technology,which was the cell fusion between the hybridoma who was HAT sensitive and can stably secrete anti-BPA monoclonal antibody and the spleen cells of the mice immunized with the antigen AE-KLH.After preparation of the hybridoma without hypoxanthine guanine phosphoribosyl transferases and immunized mice,it was successfully obtained a hybridoma named 1H4 who behaved strongly positive to BPA and KLH simultaneously.