Immunological Detection Of PCBs Residues In Chicken

Polychlorinated biphenyls（PCBs）are benzene cyclic family compounds with 209homologues.Due to stable physical and chemical properties（heat resistance,electrical insulation,oxidation,etc.）,PCBs are widely used in commercial applications such as transformers,capacitors and lubricating oils.With Improper industrial discharge,they widely dispersed into soil,air and water.They could eventually accumulate in human body and pose risk to human health through bioaccumulation and food chain enrichment.Because extremely stable in the environment,PCBs are listed as persistent organic pollutants（POPs）,but still exist widely in the environment and food.Therefore,the development of rapid,accurate and broad-spectrum detection methods for PCBs family compounds are particularly important to ensure food safety and are effective supplements to instrumental analysis.In this study,by simulating the core structure of polychlorinated biphenyls,three haptens with different chlorinated structures were designed and synthesized.Therefore,a monoclonal antibody was induced and generated which could recognize 11 homologues of PCBs.Based on the antibody materials,colloidal gold immunochromatographic detection methods and immunoassay methods with aggregation induced luminescent microspheres were developed.The results are as follows:（1）The preparation of haptens and monoclonal antibodies:Three haptens（PCBa,PCBb and PCBc）with different chlorinated structures were successfully synthesized which were confirmed by Mass spectrometry and Nuclear magnetic resonance（NMR）.Immunogen and coating antigen were prepared by coupling haptens with different carrier proteins BSA,KLH and OVA by active ester method.Broad spectrum monoclonal antibodies were obtained by immunizing BALB/c mice,cell fusion and subcloning.The results showed that mice immunized with carboxylic acid derivative hapten PCBc with PCB80 structure-liked induced a monoclonal antibody with recognition ability to PCBs.The monoclonal hybridoma cell line（C6-H8）was screened with Ig G1 subtype of antibody which could recognize 11 kinds of polychlorinated biphenyls.The identification of ELISA results showed that the IC₅₀ of 11 kinds of polychlorinated biphenyls ranged from 33.12 to 476.42 ng/ml.After obtaining the complementarity determining region（CDR）gene of antibody,the 3D model was constructed by molecular simulation and simulated docking by Auto Dock-Vina.The results showed that the intermolecular forces between the antibody and small molecules with common structure of PCBs were mainly the stacking of benzene ringπ-πbonds and halogen bonds between casein and small molecules.（2）Establishment of colloidal gold immunochromatography strip:For the purpose of rapid detection on-site,gold nanoparticles with a diameter of 20 nm were prepared by chloroauric acid reduction method.an immunochromatography method（LFIA）with colloidal gold as probe material was established using the obtained monoclonal antibody against PCBs.The results showed that the cut-off value（critical value）of PCB80,PCB169,PCB126 and PCB189 were 2-5μg/ml,and the naked eyes detection values of the other 7 PCBs were greater than 5μg/m L.（3）Establishment of Immunoassay with Aggregation Induced Emission Fluorescent Microsphere（AIEFM）:Fluorescence test strip and competitive solid-phase immunoassay were established respectively.The cut off value of PCB80 detected by fluorescent test strip is 1μg/m L.Under the optimum experimental conditions,compared with ELISA,the IC₅₀ value of 11 PCBs was increased to 6.38-252.1 ng/ml,and the detection limit（LOD）was between 0.32 and 42.15 ng/ml.The results showed that the accurate detection of spiked chicken samples could be achieved by eliminating matrix interference through dilution.The recovery was 76.90%-91.64%and the coefficient of variation was 8.5%-14.4%.In addition,the comparison with GC-MS/MS instrument method shows that the detection results of the two methods are consistent,which proves that the established method has good accuracy.In summary,the above developed methods provided a simple,broad-spectrum and sensitive tool for the detection of PCBs family compounds,and makes it possible for rapid and large-scale screening of PCBs in food.