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The ultrasensitive detection of fibroin in unearthed silk relics has great significance for investigating the origin and transmission of silk. In this study, an anti-fibroin monoclonal antibody was successfully prepared through animal immunization. Next, a label-free electrochemical immunosensor was fabricated using layer-by-layer self-assembly technology, and an indirect enzyme-linked immunosorbent assay (ELISA) was proposed. The two methods exhibited excellent sensitivity and specificity in the detection of silk fibroin, while the immunosensor showed a wider quantitative detection range (0.1-100 ng mL -1 ) and a lower detection limit (0.051 ng mL -1 ) than ELISA (10-100 ng mL -1 and 8.71 ng mL -1 ). Furthermore, the performance of the immunosensor was superior in archaeological sample detection. Taking advantage of the well-prepared monoclonal antibody, the two proposed immunological assays demonstrate tremendous potential for the ultrasensitive detection of silk fibroin, which can make great contributions to exploring the origin and transmission routes of ancient silks., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

Recently, much interest has been paid to the separation of silk produced by Bombyx mori from silk produced by other species and tracing the beginnings of silk cultivation from wild silk exploitation. In this paper, significant differences between silks from Bombyx mori and other species were found by microscopy and spectroscopy, such as morphology, secondary structure, and amino acid composition. For further accurate identification, a diagnostic antibody was designed by comparing the peptide sequences of silks produced by Bombyx mori and other species. The results of the noncompetitive indirect enzyme-linked immunosorbent assay (ELISA) indicated that the antibody that showed good sensitivity and high specificity can definitely discern silk produced by Bombyx mori from silk produced by wild species. Thus, the antibody-based immunoassay has the potential to be a powerful tool for tracing the beginnings of silk cultivation. In addition, combining the sensitive, specific, and convenient ELISA technology with other conventional methods can provide more in-depth and accurate information for species identification.

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.

We report the preparation of a specific fibroin antibody and its use for the identification of unearthed ancient silk relics. Based on the 12-amino-acid repeat sequence "GAGAGSGAGAGS", which is found in fibroin of the silkworm Bombyx mori, a specific antibody against fibroin was prepared in rabbits through peptide synthesis and carrier-protein coupling. This antibody was highly specific for fibroin found in silk. Using this antibody we have successfully identified four silk samples from different time periods. Our results reveal, for the first time, a method capable of detecting silk from a few milligrams of archaeological fabric that has been buried for thousands of years, confirming that the ancient practice of wearing silk products while praying for rebirth dated back to at least 400 BCE. This method also complements current approaches in silk detection, especially for the characterization of poorly preserved silks, promoting the investigation of silk origins and of ancient clothing cultures.

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.

A large number of Southern Song dynasty costumes were unearthed from Huangsheng tomb in Fuzhou city, Fujian province. The unlined clothing was among the funeral objects, and made of the most common leno fabric, which was light and transparent, and the clothing was in extremely poor condition. To protect the unlined clothing, scientific evaluation and protection were carried out. Three‐dimensional microscopy was used to analyse the weave structure and X‐ray fluorescence spectroscopy (XRF) and indirect enzyme‐linked immunosorbent assay (iELISA) were used to analyse scientifically the printed gold and colour paint components of the unlined clothing. The results showed that the structure of the unlined clothing was a two‐warp twisted leno, the printed gold contained Au, Cu, Fe and other elements, the coloured paint contained Pb and Hg elements, and the binding material component of the coloured paint was type I collagen. Based on the results of the analysis, the silk fibroin consolidation method was used to improve resistance to the stress and strain caused by the needle–thread repair method, and the needle–thread repair method was then used to restore the original clothing shape. This study provides a new approach for the protection of fragile silk fabrics in the future. [ABSTRACT FROM AUTHOR]

Immunomagnetic beads (IMBs) are a new immunological separation technology for protein purification, etc. In this experiment, to enrich and purify trace silk fibroin from complex soil and cultural relics, an immunomagnetic separation method was developed through coupling anti-fibroin polyclonal antibodies with magnetic beads. The key parameters were optimized to prepare the IMBs and capture the fibroin. Under optimized conditions, the capture rate with 1.2 mg of IMBs against a 100 ng/mL-1 fibroin solution was greater than 99%. The recovery rate of fibroin in soil was over 97% when the silk fibroin additive content was 10 to 50 ng, which showed that the IMB method developed in this study could successfully enrich silk fibroin in soil for indirect enzyme-linked immunosorbent assay (iELISA) detection. The results of the respective analyses of fabric and mineralized textile samples indicated that IMBs could be successfully applied for the detection of trace silk fibroin in archaeological samples. [ABSTRACT FROM AUTHOR]

The identification of ancient wool is of great importance in archaeology. Despite lots of meaningful information can be achieved by conventional detection methods, that is, light and electron microscopy, spectroscopy, and chromatography, the efficacy is likely to be limited in the detection of ancient samples with contamination or severe degradation. In this work, an immunoassay was proposed and performed for the identification of ancient wool. First, a specific antibody, which has the benefits of low cost, easy operation, and extensive applicability, was developed directly through immunizing rabbits with complete antigen (keratin). Then, an enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-labelled immunochromatographic strip (ICS) were developed to qualitatively identify the corresponding protein in ancient wool samples unearthed from Kazakhstan and China. The anti-keratin antibody exhibited high sensitivity and specificity for the identification of modern and ancient wool. The limit of detection (LOD) of the ELISA method was 10 ng/mL, and no cross-reactions with other interfering antigens have been noted. It is concluded that the immunoassays are reliable methods for the identification of ancient wool. [ABSTRACT FROM AUTHOR]

Mineralized textiles often unearthed along with metal objects, are textile residues in which the fibers are partially or completely replaced by minerals. Some textile fragments with intact fiber shapes but mineralization on the surface of a ring-shaped iron sword, had recently been found from Maoling Mausoleum in Shanxi province, China. In this study, three-dimension microscope, scanning electron microscope-energy disperse spectroscopy (SEM-EDS) and Raman spectroscopy were used to observe the morphology of fiber and analyze the mineral element information, and two immunology testing techniques would be used to determine whether the textile fragments included the silk protein. The results showed that the hilt string imprint (sample A) was a twill braiding with cinnabar remaining on the surface and the blade textile imprint (sample B) was a tabby fabric, and these fibers were mineralized by iron ions to form a cavity structure. The morphology of sample B was similar to that of mulberry silk with a smooth surface and a triangular cross section. At the same time, silk protein was successfully detected in a few milligrams of sample B using fibroin polyclonal antibody by Enzyme-linked Immunosorbent assay (ELISA) and immunofluorescence method (IFM). Thus, it can be inferred that the iron sword was covered or wrapped in silk at that time. This research provided a technical support for the identification of silk residues in mineralized textiles, which is of great significance for the study of early organic fabrics. • Mineralized samples were found on the surface of a ring-shaped iron sword. • Morphologies of mineralized fabrics were observed with microscopic techniques. • Silk protein was detected in mineralized sample using two immunological methods. [ABSTRACT FROM AUTHOR]