Sellrie F, Warsinke A, Micheel B
Department of Biotechnology, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Strasse, 14476 Potsdam-Golm, Germany.
Anal Bioanal Chem. 2006 Sep;386(2):206-10. doi: 10.1007/s00216-006-0639-3. Epub 2006 Jul 25.
This paper describes the principle of a homogeneous indirect fluorescence quenching immunoassay that uses monoclonal antibodies. It is a carrier-free assay system that is performed completely in solution. The assay system was established for the determination of a low molecular weight substance (hapten), the herbicide diuron, used as a model analyte. A fluorescein-monuron conjugate together with a fluorescence-quenching monoclonal anti-fluorescein antibody and an anti-analyte antibody (here an anti-diuron/monuron monoclonal antibody) were used as central components of the assay. The fluorescein-monuron conjugate can be bound either by the anti-fluorescein monoclonal antibody or by the anti-diuron/monuron monoclonal antibody. Due to steric hindrance, binding of both antibodies to the conjugate was not possible at the same time. By selecting the antibody concentrations appropriately, a dynamic equilibrium can be established that permits the preferential binding of the anti-diuron/monuron antibody to the conjugate, which allows the fluorescein in the conjugate to fluoresce. This equilibrium can be easily altered by adding free analyte (diuron), which competes with the conjugate to bind to the anti-diuron/monuron antibody. A reduction of anti-diuron/monuron antibody binding to the conjugate results in an increase in the binding of the anti-fluorescein antibody, which leads to a decrease in the fluorescence of the conjugate. The fluorescence is therefore a direct indicator of the state of equilibrium of the system and thus also the presence of free unconjugated analyte. The determination of an analyte based on this test principle does not require any washing steps. After the test components are mixed, the dynamic equilibrium is rapidly reached and the results can be obtained in less than 5 min by measuring the fluorescence of the fluorescein. We used this test principle for the determination of diuron, which was demonstrated for concentrations of approximately 5 nM.
本文描述了一种使用单克隆抗体的均相间接荧光猝灭免疫分析方法的原理。它是一种无载体分析系统，完全在溶液中进行。该分析系统是为测定低分子量物质（半抗原）——除草剂敌草隆而建立的，敌草隆用作模型分析物。荧光素 - 敌草隆缀合物与荧光猝灭单克隆抗荧光素抗体和抗分析物抗体（此处为抗敌草隆/敌草隆单克隆抗体）被用作该分析的核心成分。荧光素 - 敌草隆缀合物既可以与抗荧光素单克隆抗体结合，也可以与抗敌草隆/敌草隆单克隆抗体结合。由于空间位阻，两种抗体不能同时与缀合物结合。通过适当选择抗体浓度，可以建立一种动态平衡，使抗敌草隆/敌草隆抗体优先与缀合物结合，从而使缀合物中的荧光素能够发出荧光。加入游离分析物（敌草隆）可以很容易地改变这种平衡，游离分析物会与缀合物竞争结合抗敌草隆/敌草隆抗体。抗敌草隆/敌草隆抗体与缀合物结合的减少会导致抗荧光素抗体结合增加，进而导致缀合物荧光降低。因此，荧光是系统平衡状态的直接指标，也是游离未缀合分析物存在的直接指标。基于此测试原理测定分析物不需要任何洗涤步骤。测试成分混合后，能迅速达到动态平衡，通过测量荧光素的荧光，可在不到5分钟内获得结果。我们利用这种测试原理测定敌草隆，在大约5 nM的浓度下得到了验证。
