Project Objectives

The identification of organic binding media—especially in very thin layers of paintings—remains very problematic. Gas chromatography-mass spectrometry is commonly used for this purpose, but often the amount of material required for analysis is too large, or the mixture of binders too complex. The use of immunological techniques based on antibodies produced by mammals in response to invading organisms may offer an effective method for identifying binding media in extremely small samples.

Antibodies are produced by all animal immune systems in response to the presence of a foreign body or antigen. The antibody will bind to the invading microbe or injected antigen, tagging them for the attack by white blood cells. Antibody-based immunological approaches utilize the unique ability of an antibody to seek out and bind to specific regions called epitopes (which are typically short sequences of amino acids in protein antigens and short sequences of sugars in polysaccharide or glycoprotein antigens).

A commonly used technique in medical research that uses such antibodies for detection assays is the Enzyme-linked Immunosorbent Assay (ELISA). It is highly sensitive (detection limits in the order of nanograms), relatively simple, and cost-effective. Hospital and research laboratories have used ELISA successfully to identify disease in tissue samples.

GCI scientists have modified and applied this well-established technique to identify proteins in binding media such as animal glue, egg, and milk, as well as polysaccharides in plant gums. ELISA may also determine the biological source of the protein (i.e., rabbit skin glue vs. fish glue). Extensive testing demonstrates that there are no interferences from other common binding media, or with the most commonly encountered pigments. This technique can assist conservation scientists in better identifying organic materials in order to make informed treatment decisions and to improve the understanding of materials and techniques used by artists.

Project Overview

The secondary antibody is conjugated to an enzyme—in this case, alkaline phosphatase. Alkaline phosphatase catalyzes the hydrolysis of p-nitrophenyl phosphate (pNPP, which is colorless) to p-nitrophenol, a yellow dye at pH 8.5. In order to detect the antigen, pNPP is added to the ELISA plate and the pNPP solution will turn yellow (Figure 1) if the enzyme is present (this only occurs if the antigen is present). The change of the color of the solution can be measured quantitatively with a spectrophotometer or an automated plate reader. In the case of strong responses, the results can be read qualitatively by eye (Figure 2).

ELISA procedure (4pp., PDF, 78KB)

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ELISA identifies mixtures of proteins and polysaccharides in very small samples, even when the protein has suffered age deterioration. The Romano-Egyptian triptych panel from the Getty Museum (1st Century) is composed of three panels: Isis, Bearded man, and Serapsis. (Figure 3). ELISA identified animal glue and egg in a red and white paint sample from Serapis' garment. Animal glue was found in a black pigment sample from Isis' cheek. The presence of egg was due to an application of glair on selected areas of the paintings, most likely from a later restoration campaign.

Red, white, and yellow paint samples from a wooden model of the Chimu plaza, Tzchudi, dating from 800–1428 A.D., was excavated from Huaca de la Luna, Peru. (Figure 4) ELISA identified plant gum in all three of the samples. The oldest sample tested so far is a red wall painting sample from the tomb of Queen Nefertari in the Valley of the Queens in Egypt (circa 1000 BC) that tested positive for plant gums. The sample was collected by a team of scientists from Getty Conservation Institute (Corzo, 1987).

GCI scientists are currently interested in the application of antibodies to the imaging of binding media layers in paint cross sections. This will require that a completely different procedure be developed—one with the ability to both identify the proteins and to map their spatial locations.

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Page updated: November 2010