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。
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.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2013年第14期227-231,共5页
Food Science
基金
农业部公益性行业(农业)科研专项(201003008-08)
国家自然科学基金面上项目(21105030)
广州市科技计划项目(12C12101663)
关键词
克伦特罗
纯化
酶标记
ELISA
clenbuterol
purification
enzyme-labeled antibody
enzyme-linked immunosorbent assay（ELISA）
