Sandwich enzyme-linked immunosorbent assay for naringin |
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Authors: | Huihua Qu Xueqian Wang Baoping Qu Hui Kong Yue Zhang Wenchao Shan Jinjun Cheng Qingguo Wang Yan Zhao |
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Institution: | 1. Centre of Scientific Experiment, Beijing University of Chinese Medicine, China;2. School of Chinese Materia Medica, Beijing University of Chinese Medicine, China;3. School of Basic Medical Science, Beijing University of Chinese Medicine, China |
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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. |
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Keywords: | Naringin Monoclonal antibody Sandwich enzyme-linked immunosorbent assay |
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