Immunodetection of Proteins in Ancient Paint Media

Abstract

Diagnostic immunology is a powerful tool, widely used in clinical and biochemical laboratories for detecting molecules. In recent years, the technique has been adapted to materials sciences as a result of the extensive advances achieved in immunology. Today, many companies supply custom antibodies as well as new high-performance bioprobes for virtually any use. The idea of using immunodetection in the field of conservation science is not new. This analytical methodology is, in fact, particularly attractive for investigating biopolymers in painting materials; it is highly sensitive and selective with respect to the biological source of the target molecules. Among biopolymers, proteins have been widely used in the past as painting binders, adhesives, and additives in coating layers. An accurate assessment of these materials is necessary to obtain deeper insights into an artist's technique as well as to design proper restoration and conservation methods. In spite of the diagnostic potential offered by immunodetection-based techniques, some analytical drawbacks had, until recently, limited their use in routine applications in conservation science. In this Account, we highlight the most important results achieved in our research on the development of analytical methodologies based on the use of enzyme-linked immunosorbent assay (ELISA) and immuno-fluorescence microscopy (IFM) techniques for the highly sensitive and specific identification of proteins in artistic and archeological materials. ELISA and IFM offer two alternative analytical routes to this final goal: ELISA provides a fast, cost-effective, quantitative analysis of microsamples put in solution, whereas IFM combines the immunodetection of the targeted molecules with the characterization of their spatial distribution. The latter approach is of great value in the stratigraphic investigation of paintings. We discuss the limits and strengths of these methodologies in the context of the complex matrixes usually found in the investigated materials and the prolonged aging that they have undergone. Immunology is a relatively new technique in conservation science, providing a rich new field for innovation. We see two areas that are particularly ripe for future contributions. The commercial manufacture of antibodies specifically tailored for use in cultural heritage studies holds enormous potential. Moreover, the need for further refinement of detection systems in immuno-fluorescence techniques, especially the suppression of the autofluorescence background in painting materials, offers an abundance of opportunities for researchers. Immunology is a relatively new technique in conservation science, providing a rich new field for innovation.

Identification of Ancient Silk Using an Enzyme-linked Immunosorbent Assay and Immuno-fluorescence Microscopy

“…21,25 In comparison with ELISA, the IFM technique can visualize the immunoreaction in situ and simplify the pretreatments of sample. 11,20,29 Similarly, to indirect ELISA, to obtain a stronger signal, an indirect method with higher sensitivity was usually applied. In this method, a non-labeled primary antibody first binds to the target protein, and then this antibody is detected by a fluorescently labeled secondary antibody, accordingly magnifying the response signals In our previous research, ELISA was firstly used to detect proteinaceous binders e.g., collagen-I and ovalbumin, in ancient silk textiles.…”

“…In fact, immunological techniques are familiar to scientists due to their wide applications in biological research. [10][11][12][13][14][15][16][17] In recent years, immunological techniques have attracted increasing attention by those working in cultural heritage. For example, milk residues, 18,19 protein-bound materials and organic bones have been detected, [20][21][22][23][24][25] and all of these studies have demonstrated that immunoassays have the potential to identify and localize the proteins in archaeological materials both rapidly and effectively.…”

Cross-Section and Staining-Based Techniques for Investigating Organic Materials in Painted and Polychrome Works of Art: A Review

“…This phenomenon also limits the sensitivity that can be obtained using FITC. It has also been reported that antibody conjugates prepared from FITC deteriorate over time~Vagnini et al, 2008;Cartechini et al, 2010!. The fluorophore Rhodamine B is often used in biological assays, being inexpensive and robust under a variety of reaction conditions; can be covalently linked to bioactive molecules; and has suitable spectral properties in terms of absorption and fluorescence wavelength for applications in art investigation field~Arbeloa et al, Birtalan et al, 2011!. Tetramethyl Rhodamine-Isothiocyanate~TRITC!, like LISSA, reacts with primary amines and alcohols so it can stain starches and gums as well as proteins. It is dissolved in dry acetone and gives a reddish fluorescence~Wolbers, 1990, 2000!.…”

“…on cross sections was reported to be successful for recognizing casein, milk, and ovalbumin in paint layers~Vag-nini et al, 2008;Pinna et al, 2009!. The immunological approach is based on the specific interaction between the antigen~protein present in the sample! and its corresponding antibody~immunoglobulin protein!~Doménech-Carbó, 2008;Vagnini et al, 2008;Sandu et al, 2009b;Cartechini et al, 2010!. Extracted antibodies can be monoclonal or polyclonal~Wild, 2005!.…”

Fluorescence recognition of proteinaceous binders in works of art by a novel integrated system of investigation

“…Many analytical methods for the characterization of binding media (AA.VV, 2009;Mills and White, 1994;Rinuy and Gros, 1989) in polychrome works of art were developed during the past 20 years, with biochemical techniques (Cartechini et al, 2010;Dolci et al, 2008;Doménech-Carbó, 2008;Leo et al, 2009;Sandu et al, 2009) offering a useful complement to classical techniques (AA. VV, 2009;Dolci et al, 2008;Sandu et al, 2009;White, 1984) such as spectroscopy/mass spectrometry, chromatography, micro-beam, and thermoanalytical methods.…”