Bauer C G, Eremenko A V, Kühn A, Kürzinger K, Makower A, Scheller F W
Institute of Biochemistry and Molecular Physiology, University of Potsdam, Luckenwalde, Germany.
Anal Chem. 1998 Nov 1;70(21):4624-30. doi: 10.1021/ac971388s.
An amplified flow immunoassay (AFIA) was developed for cocaine, which combines a noncompetitive immunoenzymometric assay (IEMA) with an on-line detection of the enzyme label alkaline phosphatase (ALP) by a substrate-recycling biosensor. In the IEMA, the analyte cocaine first binds to a labeled polyclonal anti-cocaine antibody. Then, the excess labeled antibody is separated on an affinity column that contains a perfusion chromatography carrier modified by immobilized cocaine. The unbound complexes of the analyte cocaine with the ALP-labeled antibody are detected postcolumn. The detector senses phenol produced by ALP from phenyl phosphate. As detector, an amperometric substrate-recycling biosensor was used, which consists of a Clark-type oxygen electrode covered by tyrosinase and pyrroloquinoline quinone-dependent glucose dehydrogenase. The lower limit of detection is 380 pM (38 fmol) for cocaine. The sampling rate is 26/h. Cocaine could be detected from "real samples" with an imprecision of +/- 10% (n = 3) and with a recovery of 49 +/- 3% for various concentrations. AFIA is generally important as a new approach for the fast detection of picomolar concentrations of haptens.
开发了一种用于可卡因的放大流动免疫分析（AFIA）方法，该方法将非竞争性免疫酶测定法（IEMA）与通过底物循环生物传感器对酶标记碱性磷酸酶（ALP）进行在线检测相结合。在IEMA中，分析物可卡因首先与标记的多克隆抗可卡因抗体结合。然后，过量的标记抗体在含有固定化可卡因修饰的灌注色谱载体的亲和柱上分离。分析物可卡因与ALP标记抗体的未结合复合物在柱后进行检测。检测器检测ALP从苯基磷酸产生的苯酚。作为检测器，使用了一种安培型底物循环生物传感器，它由一个被酪氨酸酶和吡咯喹啉醌依赖性葡萄糖脱氢酶覆盖的克拉克型氧电极组成。可卡因的检测下限为380 pM（38 fmol）。采样率为每小时26次。从“实际样品”中可以检测到可卡因，不同浓度下的不精密度为±10%（n = 3），回收率为49±3%。AFIA作为一种快速检测皮摩尔浓度半抗原的新方法具有重要意义。
