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The degradation behavior of silk fibroin (SF) is a significant and intriguing subject in the area of archaeological ancient silk research. In the present study, the immunological detection techniques combined with traditional characterization methods, jointly studied the degradation process of SF from Bombyx mori (B. mori) and Antheraea pernyi (A. pernyi) through exposure to alkaline proteinase, α-chymotrypsin, pepsin, and trypsin. Spectroscopic analysis revealed that different enzymes showed similar hydrolysis effects on the secondary structure, but the changes of B. mori SF and A. pernyi SF were mainly reflected in the decrease of β-sheet and the reduction of α-helical structure, respectively. In further research of immunology, two diagnostic antibodies were prepared corresponding to SF of B. mori and A. pernyi, respectively. The enzyme-linked immunosorbent assay (ELISA) and western blot indicated the enzyme-treated SF proteins still exhibited higher immunoreactivity because the epitopes on the surface of SF molecules are retained. Although α-chymotrypsin possesses the most cleavage sites among these enzymes, the α-chymotrypsin-treated SF did not exhibit significant changes in secondary structures and high antibody binding capacity. The results deepen our understanding of the SF degradation process during enzymatic hydrolysis, and show far-reaching guiding significance in trace detection of SF.

Species identification is of key significance for exploring the origin and transmission of ancient silks. In this study, two novel methods, i.e. western blot (WB) and proteomics analyses, were proposed and established to identify the differences between silks from Bombyx mori (B. mori) and two other distinctive species (Eri silkworm and Chestnut silkworm). Three diagnostic antibodies, a polyclonal anti-silk fibroin (anti-SF) antibody (pAb), a polyclonal anti-SF-specific peptide antibody (pAsb), and a monoclonal anti-SF antibody (mAb) were designed and prepared to distinguish silk species using the antibody-based WB technique. Proteomics analysis by liquid chromatography-tandem mass spectrometry was performed to further identify silk species at the protein level. WB results indicated that the three antibodies showed high specificity and affinity and could discern B. mori silk from Eri and Chestnut silks. Biomarkers for each SF were obtained using proteomics analysis, and they have the potential to serve as standards for identifying silk species. Thus, combining WB and proteomics analyses with conventional methods can provide more accurate silk information and may be suitable for identifying other proteinaceous materials in archaeological field.

Proteinaceous materials, such as ovabumin and collagen, were commonly used as binding media, and as adhesives and protective coatings. However, the identification of ancient proteinaceous binders is a great challenge for archaeologists, due to their limited sample size, complex combinations of various ingredients and reduced availability of the binder during the process of protein degradation. In this paper, an enzyme-linked immunosorbent assay (ELISA) provides to be a particularly promising method for the detection of proteinaceous binding materials in ancient relics. The present work focused on the specific identification of proteins in archaeological binders, which was brushed on the Tripitaka. Two samples, the adhesion area (S1) and the ink area (S2), were tested by ELISA. The results showed that both S1 and S2 reacted positively when treated with an anti-collagen-I antibody. It proved the existence of proteinaceous binders in Ancient Tripitaka, and the percentage of collagen in S1 and S2 was 61.44 and 15.4%, respectively. Compared with other conventional techniques, ELISA has advantages of high specificity, sensitivity, rapidity and low cost, making it especially suitable for the protein detection in the archaeological field.

The identification of ancient silk is of great importance in both archaeology and academia. In the present work, a specific antibody having the characteristics of low cost, easy operation and extensive applicability was developed directly through immunizing rabbits with complete antigen (silk fibroin, SF). Then, antibody-based immunoassays, i.e. enzyme-linked immunosorbent assay (ELISA) and immuno-fluorescence microscopy (IFM), were established and conducted in tandem to identify the corresponding protein in ancient silks. The anti-SF antibody exhibits high sensitivity and specificity for the identification of modern and ancient silks. The detection limit of the ELISA method is about 0.1 ng/mL, and no cross-reactions with other possible interference antigens have been noted. IFM makes it possible to localize target proteins in archaeological samples, and also ensure the reliability of the ELISA results. Based on these advantages, immunological techniques have the potential to become powerful analytical tools at archaeological sites and conservation science laboratories.