title: Development of an enzyme-linked immunosorbent assay based on anti-puerarin monoclonal antibody and its applications.
abstract: An enzyme-linked immunosorbent assay (ELISA) was developed, and its application in immunoaffinity column chromatography was studied using a monoclonal antibody (MAb) against puerarin. Splenocytes isolated from a female BALB/c mouse immunised with a puerarin-bovine serum albumin (BSA) conjugate were fused with SP2/0 myeloma cells. The hybridoma cell line secreting MAb against puerarin (AA9) was acquired by screening and limiting dilution. The antibody generated was highly specific for puerarin with <0.01% cross-reactivity with over 50 structurally related chemicals, except for baicalein (51.8%). Using AA9, we developed an immunoassay for puerarin with a linear detection range of 10ng/ml to 1μg/ml. This assay system was further validated using intra- and inter-assays and recovery experiments. In addition, puerarin levels in both formulated Chinese medicines and biological samples were determined with high sensitivity and efficiency. Finally, we developed and validated protocols for knocking puerarin out of its parent medicine completely. In conclusion, we successfully developed a reliable ELISA and an immunoaffinity column for puerarin detection and knockout, which are useful tools for exploring the role of puerarin in formulated Chinese medicines.
authors.affiliation: Center of Scientific Experiment, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China.
authors.forename: Huihua
authors.initials: H
authors.lastname: Qu
authors.affiliation: School of Basic Medical Sciences, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China.
authors.forename: Guiliang
authors.initials: G
authors.lastname: Zhang
authors.affiliation: School of Basic Medical Sciences, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China.
authors.forename: Yifei
authors.initials: Y
authors.lastname: Li
authors.affiliation: School of Basic Medical Sciences, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Sun
authors.affiliation: School of Basic Medical Sciences, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Sun
authors.affiliation: School of Basic Medical Sciences, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China. Electronic address: zhaoyandr@gmail.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: School of Basic Medical Sciences, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China. Electronic address: wangqg8558@sina.com.
authors.forename: Qingguo
authors.initials: Q
authors.lastname: Wang
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D007529:Isoflavones / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D016014:Linear Models; D051379:Mice; D008807:Mice, Inbred BALB C; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D007529:Isoflavones; C033607:puerarin
keywords: ELISA; Immunoaffinity column chromatography; Monoclonal antibody; Puerarin