盐酸克伦特罗抗体纯化及直接竞争ELISA方法研究

doi:10.7506/spkx1002-6630-201314046

食品科学

盐酸克伦特罗抗体纯化及直接竞争ELISA方法研究

袁利鹏，卢倚群，孙远明，徐振林，雷红涛

1.广东农工商职业技术学院热作系，广东广州 510640；
2.广东省食品质量安全重点实验室，农业部农产品贮藏质量安全风险评价重点实验室，广东广州 510642

出版日期:2013-07-25 发布日期:2013-08-02

Purification of Anti-Clenbuterol Antibody and Development of Direct Competitive ELISA

YUAN Li-peng，LU Qi-qun，SUN Yuan-ming，XU Zhen-lin，LEI Hong-tao

1. Department of Tropical Crops, Guangdong AIB Polytechnic College, Guangzhou 510640, China；
2. Key Risk Assessment Laboratory of Agricultural Product Preservation, Ministry of Agriculture,Guangdong Provincial Key Laboratory of Food Quality and Safty, Guangzhou 510642, China

Online:2013-07-25 Published:2013-08-02

摘要/Abstract

摘要：

对比饱和硫酸铵法、辛酸法、辛酸-硫酸铵法和硫酸铵-DEAE-纤维素法4种纯化方法对盐酸克伦特罗抗体的纯化效果，研究简易过碘酸钠法与3种改良过碘酸钠法对抗体-辣根过氧化物酶标记率的影响，基于酶标记抗体建立快速检测盐酸克伦特罗的直接竞争酶联免疫分析(ELISA)方法。结果表明：辛酸法纯化抗体得率最高，但纯度最差；辛酸-硫酸铵和硫酸铵-DEAE-纤维素法纯化抗体得率较低，但纯度最好。简易过碘酸钠法所获得的抗体-酶标记得率最高，但抗体效价偏低，改良过碘酸钠法获得的抗体效价明显升高。直接竞争ELISA方法检测盐酸克伦特罗的最低检测限为2.7ng/mL，尿样加标回收率94.2%，批内变异系数为9.1%，批间变异系数为13.8%，与其他结构类似物无明显交叉反应。该方法具有操作简便、步骤少、灵敏度高、特异性更好等特点，可进一步在克伦特罗的监测中发挥重要作用。

关键词: 克伦特罗, 纯化, 酶标记, ELISA

Abstract:

Four methods, including saturated ammonium sulfate (SAS), caprylic acid (CA), caprylic acid-ammonium sulfate
(CA-AS) and ammonium sulfate-DEAE-cellulose (SAS-DEAE) were compared for their effectiveness in the purification
of anti-clenbuterol antibodies. Meanwhile, the effect of simple NaIO4 method and its three different modified versions on
antibody labeling with horseradish peroxidase (HRP) was examined. The labeled antibodies were applied to develop a direct
competitive ELISA assay for rapid detection of clenbuterol. Our results indicated that the highest yield of anti-clenbuterol
antibodies was obtained using the CA method, which, however, provided the lowest purity. The SAS-DEAE method
gave rise to the highest purity despite providing a relatively lower yield. The simple NaIO4 method was most effective
in labeling anti-clenbuterol antibodies but resulted in a lower antibody titer. By contrast, the modified NaIO4 methods
provided significantly increased antibody titer. The limit of detection of the ELISA method was 2.7 ng/mL. The recovery
of clenbuterol from spiked urine samples was 94.2%, and the inter-assay and intra-assay coefficient of variation (CV) were
13.8% and 9.1%, respectively. Moreover, no cross-reactivity with clenbuterol analogues was observed. This ELISA method
can provide a simple, sensitive and specific approach to detecting clenbuterol in animal-derived food products.

Key words: clenbuterol, purification, enzyme-labeled antibody, enzyme-linked immunosorbent assay (ELISA)

袁利鹏，卢倚群，孙远明，徐振林，雷红涛. 盐酸克伦特罗抗体纯化及直接竞争ELISA方法研究[J]. 食品科学, doi: 10.7506/spkx1002-6630-201314046.

YUAN Li-peng，LU Qi-qun，SUN Yuan-ming，XU Zhen-lin，LEI Hong-tao. Purification of Anti-Clenbuterol Antibody and Development of Direct Competitive ELISA[J]. FOOD SCIENCE, doi: 10.7506/spkx1002-6630-201314046.

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盐酸克伦特罗抗体纯化及直接竞争ELISA方法研究

Purification of Anti-Clenbuterol Antibody and Development of Direct Competitive ELISA

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