Dancheong is a traditional technique for expressing the hierarchical order of buildings and for protecting and decorating the wood surfaces. In Dancheong, wood is used as background material, pigments for coloring, and binders for the adhesion of powdered pigments to the wood’s surface (Kim et al., 2014). Animal glue was mainly used in Korean Dancheong binder, which is a kind of gelatin obtained via hot-water extraction of animal hide collagen. Animal glue has been used from the days of the Joseon dynasty, but given that acrylic emulsion which is a synthetic binder was developed in the 1970s, subsequently, this binder has been primarily used. According to the records in the Uigwe(The royal protocols of Joseon dynasty) such as “Changgyeonggung Palace Suri Dogam Uigwe” and “Gwanghaegun Sachin Chusung Dogam Uigwe”, the Dancheong binder in the Joseon dynasty made use of various organic materials such as starch paste, beeswax, lacquer and drying oil, in addition to animal glue(Koo, 2016).

Although the binder is an important factor that affects the physical characteristics and durability of the Dancheong painting film, there are limited studies on the analysis of traditional binders in Korea. In 2015, Jong Seo Park established the feasibility of the analysis of these binders by presenting pyrolysis products of various types of animal glue using pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS)(Park, 2015). Subsequent to the report, replica samples of Dancheong were fabricated using animal glue, drying oil and acrylic emulsion, and Py-GC/MS and Fourier transform-infrared spectroscopy (FT-IR) were used to determine identification factor. In the process, it was established that Py-GC/MS is a reliable method for analyzing animal glue(Park et al., 2016). In other countries, several studies have reported on the investigation of a variety of binders including plant gum, animal glue, and egg white, using GC-MS, Py-GC/MS, and FT-IR, in addition to principal component analysis. Results based on statistical analyses have also been reported(Colombini and Modugno, 2004).

The identification of animal and plant binders is possible using Py-GC/MS or FT-IR, but it is difficult to classify animal species based on only this kind of component analysis. In addition, cross-validation using a variety of assay methods is required because many different results can be obtained depending on the mixed pigment or the state of degradation. In this regard, many studies have been conducted to identify binders using enzyme-linked immunosorbent assay (ELISA) based on antigen-antibody reaction. ELISA is a quantitative analysis technique in which the analyte acts as the antigen and an enzyme is combined with an antibody to react with the antigen. The assay method was developed for the diagnosis of diseases and for clinical applications. It has been widely used in the fields of medicine, agricultural crops, and livestock. The technique can increase the sensitivity of a reaction by combing an isotope, a fluorescent substance, an enzyme, etc. with an antibody, which facilitates the identification of a sample containing the minimum amount of analyte.

In addition, extra equipment is not required apart from a microplate reader to measure absorbance. Another advantage of this approach is that it does not require a long assay time. Mazurek et al. (2008) proposed an antibody and an experimental method to analyze a variety of binders such as plant gum, animal glue, egg white, and fish glue of painting layers using ELISA(Mazurek et al., 2008). In China, Hu et al. (2012) used ELISA and immunofluorescence microscopy (IFM) to identify various binders related to animal glue, casein and fish glue in Chinese mural paintings from the 3^rd to 5^th century. Moreover, the results from quantitative analysis of the effect of mineral pigments on the denaturation of protein binders such as casein using ELISA have been reported(Ren et al., 2016).

Binders in a painting layer mixed with inorganic pigments are affected by the metal ions of the mixed pigments or undergo denaturation due to various degradation factors. As such, it is difficult to identify binders using only physicochemical component analysis. Therefore, ELISA as well as GC/MS and Py-GC/MS, are required for a more comprehensive investigation of traditional painting techniques. In this regard, this study examines the applicability of ELISA for Dancheong binder analysis based on various types of animal glues used in Korea and the Danchung samples, which are replicas of Noerok-additionally coated-Danchung. Moreover based on this investigation, we examined animal glue, an animal binder used in traditional Dancheong by applying to the technique to samples in temples of the Joseon dynasty in the 17^th and 18^th centuries.

The antibody and experimental conditions were set by referring to the work of Mazurek et al. (2008). A volume of 100 μL of the analyte and elution buffer was placed in a tube and eluted at room temperature. A volume of 200 μL of 100 mM sodium bicarbonate solution(100 mM NaHCO₃, Sigma-Aldrich, USA) was added, mixed, and allowed to stand for 10 minutes. For the elution buffer, 5 mL of 1M tris-HCl(Merck Millipore, USA) and 1 mL of 0.5M EDTA(Ethylenediaminetetraacetic acid, Merck Millipore, USA) were mixed, 180 g of urea(Merck, USA) and 25 mL of 20% SDS solution(sodium dodecyl sulfate, Sigma, USA) were added and the total volume was adjusted to 500 mL with distilled water. The pH was adjusted to 7.4 with NaOH(Duksan Science, KOR). A volume of 65 μL of sodium bicarbonate solution was added to a 96-well polystyrene plate for ELISA and 15 μL of the sample elution buffer was added and the mixture was allowed to stand at 4°C for 24 hours. The solutions were washed twice each with 300 μL of PBS(phosphate-buffered saline, Welgene, KOR) and all the samples were tested in double numbers. Blocking buffer(Sea block buffer, Pierce Chemical, USA) diluted with a ratio 1:10 (v/v) was added to PBS and washed after 60 minutes. After the addition of 80 μL of primary antibody(Collagen #AB6577, Collagen #34710, ABCAM, GBR), the solutions were left to stand at room temperature for 2 hours and then washed twice. A volume of 80 μL of secondary antibody(Rabbit IgG #AP132A, ABCAM, GBR) was added, left to stand at room temperature for 2 hours, and then washed three times. For color development, 80 μL of pNPP solution(p-nitrophenyl phosphate, Merck Milipore, USA) was added. After checking to determine whether the reaction occurred in a blank well, 80 μL of 0.75 M NaOH was added to terminate the reaction. The absorbance was measured twice at 405 nm using a microplate reader(VERSA max, Molecular Devices, USA) of MetaBio Co. Ltd, in Seoul. The absorbance threshold is the sum of the average absorbance (Aav) and 3 times the standard deviation (3 SD) of the blanks for each antibody plate. It is thought to have a positive value when the absorbance of the analyte satisfies the absorbance threshold or higher.

In this study, it was confirmed that ELISA can be used for the identification of animal glue, given that it was successfully detected in Japanese powdered animal glue, produced animal glue with various extraction conditions and replica samples mixed with pigments using ELISA method. This study is the first reported attempt to analyze animal glue, the binder in the Dancheong sample of the 17^th and 18^th Century Joseon dynasty, in which ELISA was proposed as one of the binder assay methods. It is thought that quantitative analysis of proteins that have not been confirmed in this study will be needed for more reliable analysis. In particular, further studies with an emphasis on the effects of Korean traditional pigments on the denaturation of protein binder is necessary. It is expected that a comprehensive understanding of the traditional Dancheong technique will be achieved by analyzing Dancheong patterns, pigments and the various binders that have been used in the past.

In addition, it is expected that based on the analysis of binders in various painted cultural properties as well as Dancheong, a more complete understanding of traditional painting techniques will be achieved.