Analysis of egg-based model wall paintings using an innovative combined dot-ELISA and UPLC-based approach

The chemical analysis of egg-based wall paintings – mezzo fresco technique – is an interesting topic in the field of organic binders characterisation. A revised procedure for a Dot-Enzyme-Linked ImmunoSorbent Assay (Dot-ELISA) able detect protein components of egg-based wall paintings was reported. The new Dot-ELISA procedure succeeded in maximizing the staining colour acting on the temperature during the staining reaction. Quantification of the colour intensity by visible reflectance spectroscopy allowed to obtain a straight line of the protein concentration versus the reflectance in the wavelength range 380-780 nm. The modified Dot-ELISA protocol was proposed as a semi-quantitative analytical method for the characterisation of protein binders in egg-based paintings. In order to evaluate its performance, the method was first applied to standard samples (ovalbumin, whole egg, egg white), then to model specimens, and finally on real samples (Giotto’s wall paintings). Moreover, the amino acids analysis carried out by the innovative AccQ•Tag™ Ultra Performance Liquid Chromatography was applied to both the standard and model samples and the results were compared with those of the Dot-ELISA tests. In particular, after protein hydrolysis (24h, 114°C, 6M HCl) of the samples, the amino acid derivatization by 6-aminoquinolyl-Nhydroxysuccinimidyl carbamate allowed a reproducible amino acids analysis. This UPLC AccQ•Tag™ Ultra amino acids analysis revealed itself rapid and reproducible and was applied for the first time to egg-based paintings. Since the painting technique involved the use of egg-based tempera on fresh lime-based mortars, the study enabled to investigate the influence of the alkaline environment on the egg-protein detection by both methods.