The characterization of the complex, multilayer, and multimaterial structure of a painting is fundamental for studying painting techniques and for authentication purposes. Immunological techniques taking advantage of the high specificity of antigen-antibody reactions are widely employed in bioanalytical and clinical chemistry and have also been proposed in the field of cultural heritage characterization, since they allow different proteins to be distinguished and their animal species source to be determined. In this field, fluorescence detection was employed, which is affected by aspecific autofluorescence of pigments and organic binding media. The combination of chemiluminescence (CL) imaging detection of enzyme-labeled antibodies with optical microscopy was demonstrated to allow simultaneous localization of target proteins with good spatial resolution, low background, high detectability. In this work we have developed a new ultrasensitive immunochemical procedure with CL detection for the identification and localization of two different proteins (ovalbumin and casein) present in binding media or varnishes, in small size (1-2 square mm) painting cross-sections . The immunological detection has been performed by means of specific primary antibodies, revealed by enzyme-labelled secondary antibodies and suitable CL substrates. To allow multiplexed analysis, two different enzymes were employed: horseradish peroxidase and alkaline phosphatase as labels for ovalbumin and casein, respectively. This method was optimized on standard samples, and then applied on real aged samples allowing the specific identification and localization of proteins in a paint cross-section with a resolution of 0.5 µm (i.e., within the single painting layers). In the future different organic (drying oils, resins, etc.) components could be also simultaneously detected by employing specific antibodies labelled with different CL enzymes, or bioluminescence labels.
Buragina A., Dolci L., Sciutto G., Prati S., Guardigli M., Mazzeo R., et al. (2010). Multiplexed identification and localization of proteins in painting cross-sections by chemiluminescent immunochemical microscope imaging. s.l : s.n.
Multiplexed identification and localization of proteins in painting cross-sections by chemiluminescent immunochemical microscope imaging
BURAGINA, ANGELA;DOLCI, LUISA STELLA;SCIUTTO, GIORGIA;PRATI, SILVIA;GUARDIGLI, MASSIMO;MAZZEO, ROCCO;RODA, ALDO
2010
Abstract
The characterization of the complex, multilayer, and multimaterial structure of a painting is fundamental for studying painting techniques and for authentication purposes. Immunological techniques taking advantage of the high specificity of antigen-antibody reactions are widely employed in bioanalytical and clinical chemistry and have also been proposed in the field of cultural heritage characterization, since they allow different proteins to be distinguished and their animal species source to be determined. In this field, fluorescence detection was employed, which is affected by aspecific autofluorescence of pigments and organic binding media. The combination of chemiluminescence (CL) imaging detection of enzyme-labeled antibodies with optical microscopy was demonstrated to allow simultaneous localization of target proteins with good spatial resolution, low background, high detectability. In this work we have developed a new ultrasensitive immunochemical procedure with CL detection for the identification and localization of two different proteins (ovalbumin and casein) present in binding media or varnishes, in small size (1-2 square mm) painting cross-sections . The immunological detection has been performed by means of specific primary antibodies, revealed by enzyme-labelled secondary antibodies and suitable CL substrates. To allow multiplexed analysis, two different enzymes were employed: horseradish peroxidase and alkaline phosphatase as labels for ovalbumin and casein, respectively. This method was optimized on standard samples, and then applied on real aged samples allowing the specific identification and localization of proteins in a paint cross-section with a resolution of 0.5 µm (i.e., within the single painting layers). In the future different organic (drying oils, resins, etc.) components could be also simultaneously detected by employing specific antibodies labelled with different CL enzymes, or bioluminescence labels.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
