摘要
采用酶联免疫吸附实验(Enzyme-linkedImmunosorbentAssayELISA)方法,筛查和定量检测氯霉素(Chloramphenicol)。试样经过乙酸乙酯的萃取和正己烷的净化,在温育过程中,将特异性抗体(兔抗CAP)、酶标记CAP(酶结合物)和CAP标准品或样品,加入预先包被了绵羊抗兔IgG抗体的孔内。特异性的抗体便被固定抗体所结合。与此同时,游离的CAP(存在于标准品或样品)和酶结合CAP便互相竟争CAP抗体结合部位(竞争性酶免检测)。然后温育1h洗涤除去非结合(酶标记)试剂。加入底物,结合的酶结合物可将无色的底物转化为有色产物。加入硫酸,终止底物反应。用装有450nm滤光片的酶标仪进行检测,吸光度值与样品中的CAP浓度成反比。试验结果表明,该方法在0.025～2ng/ml范围内相关系数(R2)大于0.995,检测下限0.02μg/kg,回收率为78.2%。
An enzyme-linked Immunosorbent Assay （ELISA） was established for the detection of chloramphenicol in animal tissues. After ethyl acetate extraction and mixture of n-heane clean-up, a specific antibody（rabbit anti-CAP）, enzyme labeled CAP （enzyme conjugate） and CAP standard or sample were added to the pre-coated wells followed by a single incubation step. The specific antibodies were bound by the immobillised antibodies and at the same time free CAP （present in the standard solution or sample） and enzyme conjugated CAP compete for the CAP antibody binding site （competitive enzyme immunoassay）. After an incubation time of 1 hour, the non-bound （enzyme labelled） reagents were removed in a washing step. Bound enzyme conjugate transformed the colourless chromogen into a coloured product by the addition of a chromogen substrate （TMB）. The substrate reaction was stopped by the addition of sulphuric acid. The colcour intensity was measured photometrically at 450 nm. The optical density was inversely proportional to the CAP concentration in the sample. As a result, the calibration curve should be virtually liner in the range of 0.025-2ng/ml. The limit of detection of the ELISA was 0.02μg/kg. The rate of recovery was about 78.2%.
出处
《中国农学通报》
CSCD
2006年第4期26-29,共4页
Chinese Agricultural Science Bulletin
关键词
氯霉素
ELISA
动物组织
检测
Chloramphenicol, ELISA, Animal tissues, Detection
