中药材2023，Vol.46Issue(7)：1643-1647,5.DOI:10.13863/j.issn1001-4454.2023.07.010
双抗夹心酶免疫检测法快速鉴定天花粉真伪的研究
Rapid Identification of Trichosanthis Radix by Double-Antibody Sandwich Enzyme Immunoassay
摘要
Abstract
Objective:To establish a rapid method to identify the authenticity of Trichosanthis Radix using trichosanthin(TCS)as qualitative index.Methods:New Zealand big-eared rabbits and Bal b/c female mice were immunized with TCS as immune antigen,and the rabbit serum was collected after immunization.Monoclonal antibody against TCS was prepared by hybridizing mouse myeloma cells.Polyclonal antibody and monoclonal antibody were used to establish a double-antibody sandwich enzyme immunoassay for the detection of TCS,and the reaction conditions were optimized by checkerboard method.The established method was used to measure the Trichosan-this Radix and its adulterants from different producing areas,and the ratio of the absorption value(A)to the absorption value(A0)of the blank control was used as the authenticity evaluation index.Results:The optimal reaction conditions of the established double-anti-body sandwich enzyme immunoassay were as follows:dilution ratio of rabbit serum was 500 times,concentration of monoclonal antibody was 1.0 μg/mL,concentration of enzyme-conjugate secondary antibody was 2 μg/mL.Through the detection of real samples,it was found that the absorption value of genuine Trichosanthis Radix was significantly higher than that of mixed counterfeit,and the ratio of A/A0 of genuine Trichosanthis Radix was greater than 2.0,and the ratio of A/A0 of mixed counterfeit was less than 2.0.Conclusion:The established TCS double antibody sandwich enzyme immunoassay can achieve rapid identification of authenticity of Trichosanthis Radix,simple sample processing,high detection efficiency,and has the value of application.关键词
天花粉/天花粉蛋白/双抗夹心酶免疫检测法/真伪鉴别Key words
Trichosanthis Radix/Trichosanthin/Sandwich enzyme linked immunosorbent assay/True and false identification分类
中医学引用本文复制引用
张慧婷,张雪晴,吉舒琪,李新朋,董红敬,王晓,张波..双抗夹心酶免疫检测法快速鉴定天花粉真伪的研究[J].中药材,2023,46(7):1643-1647,5.基金项目
山东省重大科技创新工程项目(2021CXGC010508) （2021CXGC010508）
临沂大学大学生创新创业训练计划项目(S202210452056) （S202210452056）
