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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.

A simple and reliable methodology to detect egg white in art samples