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.皮革制品的物种鉴定对于研究古代皮革的使用与传播具有重要意义；然而，缺乏有效的检测方法仍是一大阻碍。本研究采用鸟枪法蛋白质组学（shotgun proteomic）分析，对古代皮革制品的蛋白质组成进行鉴定。基于Swiss-Prot数据库，共鉴定出154种蛋白质。我们通过分子进化、结构域及序列比对分析对这些蛋白质进行筛选，以选出合适的靶标蛋白。最终选定Kelch样家族成员17（Kelch-like family member 17，KLHL17）与Nance-Horan综合征肌动蛋白重塑调节因子（Nance-Horan Syndrome actin remodeling regulator，NHS）用于抗体制备。通过抗体效价检测与表面等离子体共振（surface plasmon resonance，SPR）实验，测定了两种抗体的结合亲和力。此外，我们开发并优化了适用于古代皮革制品物种鉴定的酶联免疫吸附测定（enzyme-linked immunosorbent assay，ELISA）方法。两种抗体可分别特异性鉴定山羊与牛皮革样品的物种来源。本研究建立了一种兼具便携性、成本效益与高灵敏度的新型检测方法，并将其应用于皮革物种鉴定。本研究借助经典蛋白质组学方法与ELISA技术，为考古皮革制品提供了有效的检测工具。此外，本研究还为开发基于蛋白质的文物鉴定新方法提供了思路。