Hoefelschweiger Bianca K, Duerkop Axel, Wolfbeis Otto S
Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93040 Regensburg, Germany.
Anal Biochem. 2005 Sep 1;344(1):122-9. doi: 10.1016/j.ab.2005.06.030.
We report on a new and simple one-reagent method for general protein assay. It makes use of one of two new reactive labeling reagents presented here (and referred to as pyrylium [Py] labels). These can be applied for both photometric and fluorometric protein assays at near neutral pHs at room temperature. The Py labels undergo a large spectral change on conjugation to the amino group of proteins and typically change their color from blue to red. Therefore, and unlike in other assays, there is no need to separate the unconjugated (blue) label from the red conjugate, which can be determined by direct photometry with a limit of detection of 1.2 microg/ml for human serum albumin. The assay can be extended to fluorometry because the fluorescence of the free Py label is weak (with a quantum yield of <1%) but increases strongly (to >40%) on conjugation. The strong fluorescence of the red conjugates can be determined directly and without interference by the blue (and weakly fluorescent) free label. The fluorometric assay resulted in a limit of detection of 60 ng/ml for bovine serum albumin (BSA). Validation of the fluorescence assay of blood plasma samples spiked with BSA gave recoveries in the range from 91 to 103%.
我们报道了一种用于一般蛋白质测定的新型简单单试剂方法。它利用了这里介绍的两种新型反应性标记试剂之一（称为吡喃鎓[Py]标签）。这些试剂可用于在室温下接近中性pH值的光度法和荧光法蛋白质测定。Py标签与蛋白质的氨基结合时会发生很大的光谱变化，通常会从蓝色变为红色。因此，与其他测定方法不同，无需将未结合的（蓝色）标签与红色结合物分离，红色结合物可通过直接光度法测定，人血清白蛋白的检测限为1.2微克/毫升。该测定可扩展到荧光法，因为游离Py标签的荧光较弱（量子产率<1%），但结合后会强烈增加（至>40%）。红色结合物的强荧光可直接测定，不受蓝色（且荧光较弱）游离标签的干扰。荧光法测定牛血清白蛋白（BSA）的检测限为60纳克/毫升。对添加了BSA的血浆样品进行荧光测定的验证，回收率在91%至103%之间。
