Competitive flow injection enzyme immunoassay for steroids using a post-column reaction technique
Competitive flow injection enzyme immunoassay for steroids using a post-column reaction technique
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DOI：
10.1016/s0022-1759(96)00199-8
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发表时间：
1997-01-15
影响因子：
2.2
通讯作者：
Johansson, G
中科院分区：
文献类型：
--
作者：
Kronkvist, K;Lovgren, U;Johansson, G
建立了两种灵敏、快速的竞争性流动注射酶免疫分析方法，并对其在甾体类药物生物分析中的应用前景进行了评价。代替从抗体结合的半抗原-酶缀合物产生信号的更典型的方法，允许通过亲和柱的未结合级分与来自柱后反应系统中的合并通道的酶底物反应。通过电流测定法或荧光测定法检测酶产物（对氨基苯酚或4-甲基伞形酮醇）。评价了已知影响免疫测定中信号产生的几个参数，包括通过亲和柱和通过反应盘管的流速、反应盘管和亲和柱的长度。在预孵育方法中，样品在进样前与酶偶联物和抗体混合，样品通量可能高达20 h（-1）。皮质醇（0.6-80 pmol）的信号精密度约为1%（RSD），布地奈德（0.02-12.5 pmol）的信号精密度约为2%（RSD）。在皮质醇的置换测定中，当样品被注入流动中时，酶标记的分析物从亲和柱中被置换。对于12.5-1250 pmol进样，获得信号精密度为4-20%的标准曲线。在两种类型的免疫测定中使用相同的仪器设置，从而获得高度灵活的系统。需要将预孵育方法中的蛋白G亲和柱简单替换为置换试验中含有一抗的柱。
Two types of flexible, sensitive and rapid competitive flow injection enzyme immunoassay were developed and evaluated for their potential use in the bioanalysis of steroids. Instead of the more typical approach where the signal is generated from antibody-bound hapten-enzyme conjugate, the non-bound fraction passing through the affinity column was allowed to react with an enzyme substrate from a merging channel in a post-column reaction system. The enzyme product( p-aminophenol or 4-methyl umbelliferol) was amperometrically or fluorometrically detected, Several parameters known to affect signal generation in the immunoassay were evaluated, including flow rate through the affinity column and through the reaction coil, the length of the reaction coil and of the affinity column. In the pre-incubation approach, where samples were mixed with enzyme conjugate and antibodies before injection, a sample throughput as high as 20 h(-1) was possible, The signal precision was about 1% (RSD) for cortisol (0.6-80 pmol) and 2% (RSD) for budesonide (0.02-12.5 pmol). In the displacement assay for cortisol, enzyme-labelled analyte was displaced from the affinity column when the sample was injected into the flow. A standard curve was obtained with a signal precision of 4-20% for 12.5-1250 pmol injected. The same instrumental set-up was used in both types of immunoassay, and thus a highly flexible system was obtained. A simple replacement of the affinity column from protein G in the pre-incubation approach to a column containing primary antibodies in the displacement assay was needed.