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Anal Bioanal Chem. 2016 Jan;408(1):203-15. doi: 10.1007/s00216-015-9089-0. Epub 2015 Oct 15.

Influence of pigments and protein aging on protein identification in historically representative casein-based paints using enzyme-linked immunosorbent assay.

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Department of Scientific Research, The Metropolitan Museum of Art, 1000 5th Ave, New York, NY, 1128, USA.
Genzyme Science Center, 49 New York Avenue, Framingham, MA, 01701, USA.
Art Conservation, University of Delaware, 303 Old College, Newark, DE, 19716, USA.
College of Physicians and Surgeons, Columbia University, 630 W 168th St, New York, NY, 10032, USA.
Department of Scientific Research, The Metropolitan Museum of Art, 1000 5th Ave, New York, NY, 1128, USA.


A systematic study on the influence of pigments and sample aging on casein identification was performed on 30 reconstructed paints. The protein in all the paints was extracted into solution for analysis. The amount of protein that can be retrieved for solution-based analysis in each of the reconstructed paints was studied with a well-developed NanoOrange method before and after artificial aging. The results showed that in the paints with calcium phosphate (in bone black) and copper carbonate, hydroxide, or acetate (in verdigris and azurite), the amount of protein that can be retrieved for liquid-phase analysis is much smaller than the other paints, indicating that the protein degradation was accelerated significantly in those paints. Carbon (in vine black), calcium carbonate (in natural chalk), and calcium sulfate (terra alba gypsum and ground alabaster) did not affect much the amount of protein that can be retrieved in the paints compared to non-pigmented binder, meaning that the protein degradation rate was not affected much by those pigments. Artificial aging was observed to decrease the amount of retrievable protein in all the reconstructed paints that were studied. The enzyme-linked immunosorbent assay (ELISA) method was applied to the 28 reconstructed paints (except two verdigris paints) to assess the protein identification. The ELISA responses from the different paints were compared at fixed protein concentrations. Natural chalk, bone black, raw sienna, stack lead white, and cochineal red-violet lake had the lowest ELISA signal in this study, which indicated that the binding sites (epitopes) on the target protein in these paints are likely to deteriorate more than those in the other paints. Artificial aging did not influence the ELISA response as much as the pigments when the protein concentration was kept the same for the paints that were studied.


Casein paint; Cultural heritage; ELISA; NanoOrange; Pigment and protein interaction; Protein degradation

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