Dipartimento di Scienze per gli Alimenti, la Nutrizione e l'Ambiente, Universita degli Studi di Milano, Milano, Italy.
J Biosci. 2013 Jun;38(2):397-408. doi: 10.1007/s12038-013-9321-z.
A protocol for a simple and reliable dot-blot immunoassay was developed and optimized to test work of art samples for the presence of specific proteinaceus material (i.e. ovalbumin-based). The analytical protocol has been extensively set up with respect, among the other, to protein extraction conditions, to densitometric analysis and to the colorimetric reaction conditions. Feasibility evaluation demonstrated that a commercial scanner and a free image analysis software can be used for the data acquisition and elaboration, thus facilitating the application of the proposed protocol to commonly equipped laboratories and to laboratories of museums and conservation centres. The introduction of method of standard additions in the analysis of fresh and artificially aged laboratory-prepared samples, containing egg white and various pigments, allowed us to evaluate the matrix effect and the effect of sample aging and to generate threshold density values useful for the detection of ovalbumin in samples from ancient works of art. The efficacy of the developed dot-blot immunoassay was proved testing microsamples from 13th-16th century mural paintings of Saint Francesco Church in Lodi (Italy). Despite the aging, the altered conditions of conservation, the complex matrix, and the micro-size of samples, the presence of ovalbumin was detected in all those mural painting samples where mass-spectrometry-based proteomic analysis unambiguously detected ovalbumin peptides.
建立并优化了一种简单可靠的斑点印迹免疫检测法的方案，用于检测艺术品样本中是否存在特定的蛋白质材料（例如基于卵清蛋白的物质）。该分析方案在蛋白提取条件、密度分析和比色反应条件等方面进行了广泛的设置。可行性评估表明，商用扫描仪和免费的图像分析软件可用于数据采集和处理，从而促进了该方案在常规配备的实验室、博物馆和保护中心实验室中的应用。在分析新鲜和人工老化的实验室制备样品（包含蛋清和各种颜料）时，引入标准添加法，可以评估基质效应和样品老化的影响，并生成用于检测古代艺术品样本中卵清蛋白的有用阈值密度值。该斑点印迹免疫检测法的有效性通过测试来自洛迪（意大利）圣弗朗西斯科教堂 13 至 16 世纪壁画的微样本得到了证明。尽管存在老化、保存条件改变、基质复杂以及样本微小等问题，但在所有那些壁画样本中均检测到了卵清蛋白的存在，而基于质谱的蛋白质组学分析明确检测到了卵清蛋白肽。
