Development Of Rapid Immunoassays Of Anti-nutritional Factors,potential Hazardous Substances Of Traditional Chinese Medicine And Heavy Metals

With the rapid development of economy,people's income is getting higher and higher,and their awareness of food safety and health is getting higher and higher.In this study,we focuse on anti-nutritional factors,potentially harmful substances in traditional Chinese medicine and heavy metals,and the monoclonal antibodies against soybean trypsin inhibitor,oxazolidinthione,colchicine,aconitine,abietic acid,aristolochic acid,lead ion and mercury ion were prepared,and based on these antibodies,the corresponding rapid immunological detection methods have been established.1.Preparation of anti-nutritional factor monoclonal antibodies and establishment of immunological detection methodsThrough immunization of mice and cell fusion,the paired antibodies 3E9 and 2F12which can recognize both BBI and KTI were obtained.Under the optimized conditions of double-antibody sandwich ELISA,the detection limits of BBI and KTI were 0.7 ng/mL and2.2 ng/mL,respectively.And based on these antibodies,colloidal gold immunochromatographic test strips for the detection of KTI and BBI were established respectively,and the detection limit for BBI and KTI were 10 ng/mL and 50 ng/mL,respectively,and soybean milk samples were tested.The hapten of oxazolidinethione was designed,and the monoclonal antibody 3B4 of oxazolidinethione was prepared.Its subtype is IgG1 and the affinity constant is 3.78�10⁹L/mol.Under the optimized conditions of ic-ELISA,and the IC₅₀ of 3B4 was 541.72 ng/mL,the detection interval was 95.33?3078.24 ng/mL,there is no cross-reaction with its structural analogs.The colloidal gold immunochromatographic test strip for detection of oxazolidinethione was established and optimized,and the visual detection interval in PBS was6?14?g/mL,and the visual detection interval in rapeseed samples was 40?100?g/mL.The analog of colchicine was selected as the hapten,and it was used to synthesize the coating antigen and immunogen,the antibody 1E4 was prepared,and its subtype is IgM.The ic-ELISA was optimized,and the IC₅₀ and detection interval were 0.43 ng/mL and 0.09?2.16ng/mL,respectively,and there was no cross-reaction with structural analogs.A quantitative colloidal gold immunochromatography test strip for detection of colchicine was established,and the visual detection interval in PBS was 0.5?10 ng/mL,and the recovery rate was95.3?99.54%;the visual detection interval in milk was 1?25 ng/mL,and the recovery rate was 93.56-97.4%;the visual detection interval in beef was 2.5?50 ng/mL,and the recovery rate was 84.96-95.8%;.the visual detection interval in edible lily was 1?25 ng/mL ng/mL,and the recovery rate was 93.97?102.33%;the visual detection interval in daylily was 2.5?25ng/mL,and the recovery rate was 83.51?106.52%.2.Preparation of monoclonal antibodies for potentially hazardous substances in traditional Chinese medicine and establishment of immunological detection methodsThe hapten of aconitine was designed and the monoclonal antibody 5D1 was prepared,its subtype was IgG1 and the affinity constant is 1.25�10¹⁰L/mol.Under the optimized conditions of ic-ELISA,and the IC₅₀ of 5D1 was 3.45 ng/mL,the detection interval was1?11.91 ng/mL,and there is no cross-reaction with its structural analogs.The recoveries of Chuanmutong and Fuzilizhong pills were 91.2?106.88%and 93.02?103.08%,respectively.The colloidal gold immunochromatographic test strip for detection of aconitine was established and optimized,and the visual detection interval in PBS was 5?10 ng/mL;the visual detection interval in Chuanmutong samples was 10?25 ng/mL;the visual detection interval in Fuzilizhong pills was 100?250 ng/mL.The protein was sulfhylated and then coupled with the double bond of abietic acid to synthesize the coating antigen and immunogen.The antibody 3H8 was prepared,and its subtype was IgM,the affinity constant was 7.93�10⁸L/mol.Under the optimized conditions of ic-ELISA,and the IC₅₀ and detection interval of 3H8 were 15.17 ng/mL and 4.23?54.43ng/mL,respectively,there is no cross-reaction with its structural analogs,and the recoveries of Dieda tablets and Arthritis tablets were 91.67?101.83%and 92.48?101.14%,respectively.The visual detection interval of colloidal gold immunochromatographic strip was 50?500ng/mL in PBS and 100?1000 ng/mL in Dieda tablets and Arthritis tablets.By synthesizing the coating antigen and immunogen of aristolochic acid,the antibody2A8 was prepared,its subtype was IgG1,and the affinity constant is 1.96�10⁸L/mol.Under the optimized conditions of ic-ELISA,the IC₅₀ of 2A8 was 4.21 ng/mL and the detection interval was 0.62?28.66 ng/mL,and there was no cross-reaction with its structural analogs,and the recovery rate in Chuanmutong was 95.48?101.89%.The colloidal gold immunochromatographic test strip was established and optimized.The visual detection interval in PBS was 10?100 ng/mL,and the visual detection interval in Chuanmutong sample was 250?500 ng/mL.3.Preparation of heavy metal monoclonal antibodies and establishment of immunological detection methodsThe coating antigen and immunogen of lead ion were synthesized by the bifunctional chelating agent ITCBE,and the antibody 2C3 was prepared,its subtype was IgG2a,and the affinity constant is 2.38�10¹⁰L/mol.Under the optimized conditions of ic-ELISA,The IC₅₀and the detection interval of were 0.51 ng/mL and 0.15?1.75 ng/mL,respectively,and there was no cross-reaction with other metal ions,the recovery rate in brown rice was91.16?104.27%.The fluorescence immunochromatographic quantitative detection test strip was establish and optimized.The visual detection interval in HBS was 5?25 ng/mL;the detection interval in brown rice was 10?50 ng/mL,and the recovery rate is 93.25?111.24%.The coating antigen and immunogen of mercury ion were synthesized by the bifunctional chelating agent ITCBE,and the antibody 2C10 was prepared,its subtype is IgG1,and the affinity constant is 5.67�10⁹L/mol.Under the optimized conditions of ic-ELISA,The IC₅₀and the detection interval of were 2.69 ng/mL and 0.44?16.25 ng/mL,respectively,and there was no cross-reaction with other metal ions,the recovery rate in milk sample was94.06?101.58%.The colloidal gold immunochromatographic detection test strip was established and optimized.The visual detection interval in HBS is 10?50 ng/mL,and the visual detection interval in milk samples is 20?100 ng/mL.