孔雀石绿单克隆抗体制备和酶联免疫检测方法的建立 |
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引用本文: | 王宇,杨金易,徐振林,戚平,沈玉栋.孔雀石绿单克隆抗体制备和酶联免疫检测方法的建立[J].分析化学,2016(9):1385-1393. |
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作者姓名: | 王宇 杨金易 徐振林 戚平 沈玉栋 |
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作者单位: | 1. 广州市食品检验所,广州,510410;2. 华南农业大学食品学院,广州,510640 |
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基金项目: | 国家自然科学基金(31371769),广东省科技计划项目(.2014A020219008;2016A040403077),广东省自然科学基金项目(Nos.2013030013338;2014A030306026)项目资助。 |
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摘 要: | 针对孔雀石绿三苯环特征结构,设计合成1种高质量半抗原。通过偶联载体蛋白、动物免疫和细胞融合,制备出一株高质量的单克隆抗体MG-DA4-C7,亚类为IgG1,轻链为κ型。通过对包被原浓度、抗体浓度、二抗浓度的优化,建立了孔雀石绿间接竞争酶联免疫分析方法。方法半抑制浓度( IC50)为0.96μg/L,线性检测范围(IC20~C80)为0.1~8.1μg/L,LOD(IC10)为0.05μg/L,回归方程y=-0.3274lgx+0.4698(R2=0.9891)。本方法特异性高,与代谢物隐孔雀石绿交叉反应小于0.1%,与结晶紫、灿烂绿交叉反应率分别为18.1%和26.5%;实际样品添加回收率为87.3%~107.3%。本方法测定结果经HPLC-MS/MS方法确证,二者相关系数达0.999。本方法可用于鱼类等水产品中孔雀石绿残留的实际检测。
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关 键 词: | 孔雀石绿 半抗原 单克隆抗体 抗原识别 酶联免疫分析 |
Synthesis of Novel Hapten and Development of Monoclonal Antibody-based Enzyme-Linked Immunosorbent Assay for Malachite Green in Fish Samples |
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Abstract: | To produce specific antibodies against malachite green ( MG) , one special hapten was synthesized and characterized, and conjugated to carrier protein as immunogen. The immunogen showed excellent reactogenicity and immunogenicity. One specific monoclonal antibody (mAb, named MG-DA4-C7) with high sensitivity and specificity for MG in indirect competitive enzyme-linked immunoassay ( icELISA ) was screened. The isotype was IgG1 and the light chain was κ type. After optimization of ELISA conditions, the proposed icELISA showed a 50% inhibition value ( IC50 ) of 0. 96 μg/L, a linear range ( IC20-IC80 ) of 0. 1-8. 1 μg/L and a limit of detection ( LOD, IC10 ) of 0. 05 μg/L for determination of MG. The assay showed cross-reactivity of 18. 1%, 26. 5% with crystal violet and brilliant green, respectively, and negligible cross-reactivity with other metabolites of MG (<0 . 1%) . The average recoveries of MG from spiked fish samples were from 87. 3% to 107. 3%. Good correlation (R2=0. 999) was obtained between the results of icELISA and those of liquid chromatography-tandem mass spectrometry analysis. The proposed icELISA is suitable for the determination of MG in fish samples in a simple and sensitive manner. |
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Keywords: | Malachite green Hapten Monoclonal antibody Antigenic-determinant Enzyme-linked immunoassay |
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