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

Abstract

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.

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

“…Therefore, the dilution ratios of 1:3000 for the anti-SF primary antibody and 1:5000 for the secondary were considered to be the optimal antibody dilutions in view of the compromise between high sensitivity and low background. 22 Prior to sample detection, a series of controls, such as positive control (silk fibroin), negative control, BSA and PBS control, were set up to determine the threshold value. In order to ensure the accuracy of the results, the S/N ratio method was employed in this study.…”

“…[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. However, most of the antibodies used in these immunoassays are readily commercially available "off-theshelf", and the need for tailored antibodies for targeted cultural heritages with high sensitivity and specificity is increasingly desirable.…”

“…22,26,27 These features make ELISA a promising method in conservation science. To ensure greater sensitivity and specificity, an indirect ELISA approach is usually used.…”

Miniaturized Biosensors to Preserve and Monitor Cultural Heritage: from Medical to Conservation Diagnosis

“…TheC L system showed two orders of magnitude lower LODs than the colorimetric system. [9,11] Our device, which has been developed for on-site application, showed performance similar to those of standard laboratory immunological methods.F urthermore,t he amount of sample (0.5 mg) is similar to the amounts used in other ELISA approaches,which range from 100 mgto1mg. [10] TheL ODs displayed by the CL system were close to those (that is,afew nanograms of target protein) of other ELISA laboratory methods based on fluorescence detection.…”

“…[7] In fact, although originally developed for medicine,i mmunoassays have been used to detect proteins in paintings for over 45 years. [10][11][12] Past research has also confirmed the absence of detectable interferences owing to paint materials. Previous studies have supported the suitability of the immunochemical analyses for aged and historical samples, showing that aging does not hamper recognition of proteins by immunological methods.…”