Preparation Of Monoclonal Antibody Against Bisphenol A And Establishment Of Its Gold-immunochromatography

Bisphenol A (bisphenol A, BPA) is one kind of phenolic substances which were synthetized by two molecular phenol and one molecular acetone in acid conditions. Its structure is similar with the natural hormones17-β estrogen and synthetic hormenes, which has been identified as one kind of exogenous estrogen, for one of the EDCs. Bisphenol A is an important raw material for the PC. BPA is contained in many daily consumer goods such as food packaging containers and articles for children including baby bottles. BPA can be released into the food or drinks when it get heating. In the past two years, the problems caused by BPA get the people more and more attention.Bisphenol A hapten (BPAH) was prepared by diazotization, and then Mixture anhydrides method and Carbodiimide method were used for coupling BPAH with BSA/OVA to prepare immunogen/coating antigen of BPA respectively. Spleen cells from BALB/c mice which was immune by the artificial antigens were fused with SP2/0cells to produce hybridomas. Three times of subcloning were carried out by limited dilution then the cell lines which can secrete antibodies against BPA were piceked out. Octylic acid-ammonium sulfate precipitation method was use to purify the McAbs from the ascites which were induced by hybridomas in vivo. Then SDS-PAGE and ultraviolet spectrophotometry were used to analysis the purity and content of the antibody. Colloidal gold were prepared by citrate-natrium method. After charactered by TEM, colloidal gold were used to mark the McAbs against BPA. At last antibody-colloidal gold compounds were made for test strip preparation.Results:(1)The immunogen and coating antigen were synthesized successfully and identified by UV scanning spectrum and SDS-PAGE. The coupling ration of BPAH to BSA and OVA were11:1and6.8:1respectively.(2)The monoclonal antibody is identified as IgG2b subtype and κ light chain.(3)The linear regression line equation is y=15.851x-4.0353, linear correlation coefficient R2=0.9903,and the linear detecting the range of calibration curve for BPA was64ng/ml to8192ng/ml, IC50=2567.22ng/ml.(4)Cross reaction results show that antibodies against BPA has good specificity, the analogues’ crossover rate were being found lower than0.01%, excepted the double phenolic acids, which is9.6%.(5)Colloidal gold were prepared by citrate-natrium method. To establish the GICA,McAb against BPA conjugated to colloidal gold served as the analysis probe. Artificial antigen, BPAH-OVA acted as the coating antigen(test line), and goat anti-mouse IgG acted as the control dot(control line)were immobilized on the membrane. The optimal diameter of colloidal gold particles was about40nm. The optimal concentration of the coating antigen was0.2μg/ml, the goat anti-mouse IgG was0.2mg/ml.(6)The sensitivity of GICA is100ng/ml, detection time was10min, and without any cross reactions to other kind of structural analogues while good reproducibility. Conclusion:the McAb against BPA is specific and the GICA will be a convenient, quick and effective detection method for detecting BPA.