The Preparation Of Monoclonal Antibody For The Development Of Fluorescence Polarization Immunoassay For Bisphenol A

Bisphenol A (BPA) belongs to environmental hormone and affects human safety and health. BPA also can lead to premature infants, abortion, proatate cancer or affect human ecdocrine system, destroy DNA and so on. Yet, BPA is an important and widely used organic chemical raw material. During the production and using of BPA, BPA may migrate into environment, then it would influence human health. In order to protect the safety of the ecosystem and human health, the establishment of rapid detection of BPA is particularly important.In this paper, according to the particular structure of bisphenol A, 4,4-bis(4-hydroxyphenyl)-valeric acid (BVA) was selected as the hapten to couple with bovine serum albumin and the conjugate (BVA-BSA) was used as the immunogen for the monoclonal antibody production. Balb/c mice were injected with BVA-BSA to obtain immunized spleen cells fused with SP2/0 cells. Positive hybridoma cells were obtained through indirect ELISA screening and cells subcloning. Monoclonal antibody against BPA was generated by intraperitoneal injected Balb/c mice to prepare ascites. The monoclonal antibody was purified by acid-ammonium sulfate and identified by SDS-PAGE and ic-ELISA. The monoclonal antibody with good sensitivity and high specificity was used for the analysis of environmental samples.The synthesis of fluorescent tracers were coupled BVA with fluoresceins. In this study, three fluoresceins (lysFITC, EDF, AMF) were selected to prepared fluorescent marker. EDF and lysFITC were derived from FITC. Fluorescent tracers were produced from fluoresceins with activated hapten. The fluorescent tracers were purified through TLC procedure and identificated by fluorescence polarization immunoassay (FPIA). BVA-AMF was found to be the best fluorescent tracer to establish the method FPIA.Based on a new monoclonal antibody and a good fluorescent tracer, FPIA was established, with a detection limit of 5.6ng/mL, IC50 of 140.6ng/mL, the linear range of 11.32-904.21ng/mL, the correlation coefficient R2=0.9913. Compared with ELISA and HPLC methods, FPIA showed reliability and high correlation with ELISA of 0.9964 and HPLC of 0.9971. For the recoveries, the spiked recoveries ranged from 87.91-114.28% by FPIA and from 85.40-104.18% by HPLC, while recoveries of ELISA were 92.60-112.70%. The experimental results indicated fluorescence polarization immunoassay method was reliable for the analysis of sample for bisphenol A residual.