title: Sandwich enzyme-linked immunosorbent assay for naringin.
abstract: Among the currently used immunoassay techniques, sandwich ELISA exhibits higher specificity, lower cross-reactivity, and a wider working range compared to the corresponding competitive assays. However, it is difficult to obtain a pair of antibodies that can simultaneously bind to two epitopes of a molecule with a molecular weight of less than 1000 Da. Naringin (Nar) is a flavonoid with a molecular mass of 580 Da. The main aim of this study was to develop a sandwich ELISA for detecting Nar. Two hybridomas secreting anti-Nar monoclonal antibodies (mAbs) were produced by fusing splenocytes from a mouse immunised against Nar-bovine serum albumin (BSA) conjugated with a hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line; a sandwich ELISA for detecting Nar was developed using these two well-characterised anti-Nar mAbs. The performance of the sandwich assay was further evaluated by limit of detection (LOD), limit of quantification (LOQ), recovery, and interference analyses. A dose-response curve to Nar was obtained with an LOD of 6.78 ng mL(-1) and an LOQ of 13.47 ng mL(-1). The inter-assay and intra-assay coefficients of variation were 4.32% and 7.48%, respectively. The recovery rate of Nar from concentrated Fructus aurantii granules was 83.63%. A high correlation was obtained between HPLC and sandwich ELISA. These results demonstrate that the sandwich ELISA method has higher specificity for Nar than indirect competitive ELISA.
authors.affiliation: Centre of Scientific Experiment, Beijing University of Chinese Medicine, China.
authors.forename: Huihua
authors.initials: H
authors.lastname: Qu
authors.affiliation: School of Basic Medical Science, Beijing University of Chinese Medicine, China.
authors.forename: Xueqian
authors.initials: X
authors.lastname: Wang
authors.affiliation: School of Basic Medical Science, Beijing University of Chinese Medicine, China.
authors.forename: Baoping
authors.initials: B
authors.lastname: Qu
authors.affiliation: School of Basic Medical Science, Beijing University of Chinese Medicine, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Kong
authors.affiliation: School of Chinese Materia Medica, Beijing University of Chinese Medicine, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: School of Chinese Materia Medica, Beijing University of Chinese Medicine, China.
authors.forename: Wenchao
authors.initials: W
authors.lastname: Shan
authors.affiliation: School of Chinese Materia Medica, Beijing University of Chinese Medicine, China.
authors.forename: Jinjun
authors.initials: J
authors.lastname: Cheng
authors.affiliation: School of Basic Medical Science, Beijing University of Chinese Medicine, China. Electronic address: wangqg8558@sina.com.
authors.forename: Qingguo
authors.initials: Q
authors.lastname: Wang
authors.affiliation: School of Basic Medical Science, Beijing University of Chinese Medicine, China. Electronic address: zhaoyandr@gmail.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhao
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002957:Citrus / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D044950:Flavanones / Q000032:analysis*
chemical_list: D044950:Flavanones; C005274:naringin
keywords: Monoclonal antibody; Naringin; Sandwich enzyme-linked immunosorbent assay