Development of an analytical protocol for a fast, sensitive and specific protein recognition in paintings by enzyme-linked immunosorbent assay (ELISA)

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 beta-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.