Rapid Identification of Ancient Leather Species Using an Enzyme-Linked Immunosorbent Assay Based on Proteomic and Evolutionary Analyses

The species identification of leather artifacts is of great significance for studying the use and spread of ancient leathers; however, the absence of effective detection methods remains an obstacle. Here, we performed a shotgun proteomic analysis to identify the protein composition of ancient leather artifacts. Based on the Swiss-Prot database, 154 proteins were identified. We investigated these proteins using molecular evolution, structural domain, and sequence alignment analyses to select suitable proteins. Two proteins, Kelch-like family member 17 (KLHL17) and Nance-Horan Syndrome actin remodeling regulator (NHS), were selected for antibody preparation. Their binding affinities were determined by antibody potency and surface plasmon resonance (SPR). Furthermore, we developed and optimized an enzyme-linked immunosorbent assay (ELISA) suitable for the species identification of ancient leather artifacts. Two antibodies specifically identified the species of leather samples from goats and cattle, respectively. We established a new method with the advantages of portability, cost-effectiveness, and high sensitivity that was applied to leather species identification. Our study provides an effective detection tool for archeological leather artifacts utilizing the classical proteomics approach and ELISA technique. In addition, this study provides insights into the development of new protein-based methods for the identification of cultural relics.