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We report the development of an indirect ELISA procedure for specific identification of chicken-egg yolk and animal glues in painting micro-samples. The results presented integrate previously published work on ELISA recognition of bovine β-casein and chicken ovalbumin in painting materials. The integrated final ELISA procedure-optimised for protein extraction, immuno-reagent concentrations, blocking solution, incubation time, and temperature-enables multiplex identification, in single samples, of proteinaceous materials, i.e. chicken-egg yolk and albumen, animal glues, and bovine milk and/or casein, mainly used by painters in the past. The procedure has been systematically tested on laboratory models of mural and easel paintings, both naturally and artificially aged, to assess possible inhibitory effects on the immuno-reaction caused by inorganic painting materials (pigments and substrates) and by protein degradation resulting from aging processes. Real samples from case studies, which had previously been investigated and characterised by spectroscopy and chromatography, were successfully studied by use of the developed ELISA procedure. The commercial availability of all the immuno-reagents used, the affordable analytical equipment, and the specificity, sensitivity, and rapidity of ELISA make this method very attractive to diagnostic laboratories in the field of cultural heritage science. Possible further developments to the analytical potential of this technique include improvement of antibody performance and inclusion of other classes of bio-molecules as analytical targets.

Enzyme-linked immunosorbent assay (ELISA) analysis of proteins offers a particularly promising approach for investigations in cultural heritage on account of its appreciated properties of being highly specific, sensitive, relatively fast, and cost-affordable with respect to other conventional techniques. In spite of that, it has never been fully exploited for routine analyses of painting materials in consideration of several analytical issues that inhibited its diffusion in conservation science: limited sample dimensions, decrease of binder solubility and reduced availability of antibody bonding sites occurring with protein degradation. In this study, an ELISA analytical protocol suited for the identification of aged denatured proteins in ancient painting micro-samples has been developed. We focused on the detection of bovine β-casein and chicken ovalbumin as markers of bovine milk (or casein) and chicken albumen, respectively. A systematic experimentation of the ELISA protocol has been carried out on mock-ups of mural and easel painting prepared with 13 different pigments to assess limits and strengths of the method when applied for the identification of proteins in presence of a predominant inorganic matrix. The analytical procedure has been optimized with respect to protein extraction, antibodies' concentrations, incubation time and temperature; it allows the detection of the investigated proteins with sensitivity down to nanograms. The optimized protocol was then tested on artificially aged painting models. Analytical results were very encouraging and demonstrated that ELISA allows for protein analysis also in degraded painting samples. To address the feasibility of the developed ELISA methodology, we positively investigated real painting samples and results have been cross-validated by gas chromatography-mass spectrometry.

In situ, non-invasive investigations by means of portable X-ray fluorescence and fibre optic reflectance mid-infrared (mid-FTIR) spectroscopy of painted Böttger Stoneware objects have been carried out through the MOLAB transnational access to the Porcelain Collection of the Staatliche Kunstsammlungen in Dresden. It has been possible to gather information regarding the composition of the black glaze by applying a principal component analysis to the elemental analysis to distinguish between the variations of lead, iron and manganese compositions of each glaze. It has been furthermore feasible to combine molecular spectroscopy for characterization of the constituent painting materials, namely lead white as cerusite and hydrocerusite, the use of cinnabar, azurite and Prussian blue leading to a better knowledge of the state of conservation and utility of certain pigments that may give rise to chronology of the decorative artwork. The identification of oxalates namely whedellite and moolooite are assigned as degradation products relative to the decorative areas.

The palette used in two paintings by Paul Cézanne, L'étang des soeurs dated c. 1875 and La route tournante, made in the last year of his life (1902), were analyzed using non-invasive spectroscopic methods. X-ray fluorescence combined with principal components analysis (PCA) and supported by reflectance near- and mid-FTIR was shown to be a powerful analytical tool to draw conclusions about the chemical identification of inorganic materials in paintings. Pigments and fillers such us Thénard's blue, Prussian blue, red ochre, kaolin, vermilion, lead white, zinc white and barium sulphate, were identified. Evidence for three different pigments, namely a copper arsenite pigment, chrome green (a mixture of chrome yellow and Prussian blue) and viridian has been obtained by the PCA analysis of elemental compositions of green hues.