Analysis of egg-based model wall paintings by use of an innovative combined dot-ELISA and UPLC-based approach
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The chemical analysis of egg-based wall paintings—the mezzo fresco technique—is an interesting topic in the characterisation of organic binders. A revised procedure for a dot-enzyme-linked immunosorbent assay (dot-ELISA) able to detect protein components of egg-based wall paintings is reported. In the new dot-ELISA procedure we succeeded in maximizing the staining colour by adjusting the temperature during the staining reaction. Quantification of the colour intensity by visible reflectance spectroscopy resulted in a straight line plot of protein concentration against reflectance in the wavelength range 380–780 nm. The modified dot-ELISA procedure is proposed as a semi-quantitative analytical method for characterisation of protein binders in egg-based paintings. To evaluate its performance, the method was first applied to standard samples (ovalbumin, whole egg, egg white), then to model specimens, and finally to real samples (Giotto’s wall paintings). Moreover, amino acid analysis performed by innovative ultra-performance liquid chromatography was applied both to standards and to model samples and the results were compared with those from the dot-ELISA tests. In particular, after protein hydrolysis (24 h, 114 °C, 6 mol L−1 HCl) of the samples, amino acid derivatization by use of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate enabled reproducible analysis of amino acids. This UPLC amino acid analysis was rapid and reproducible and was applied for the first time to egg-based paintings. Because the painting technique involved the use of egg-based tempera on fresh lime-based mortar, the study enabled investigation of the effect of the alkaline environment on egg-protein detection by both methods.
KeywordsUPLC-based amino acid analysis dot-ELISA Egg-based wall paintings Cultural heritage conservation
The authors express their gratitude to Professor Guido Botticelli, for careful preparation of the model samples and continuous exchange of opinions during the entire study, and to Dr Mirella Baldan, R. & C. Scientifica s.r.l., for fruitful discussions on the dot-ELISA tests. Thanks are due also to the Opificio delle Pietre Dure team for cooperation during study of the Giotto sample. Financial support from Università degli Studi di Firenze (Fondi d’Ateneo ex-60 %), Ente Cassa di Risparmio di Firenze, and from Regione Toscana PAR-FAS, SICAMOR PROJECT are gratefully acknowledged. MP thanks Regione Toscana, Italy, TemArt Project European Fund for Regional Development (POR CreO FESR 2007–2013) and the Project Partner Adarte snc, for financial support.