Discerning Silk Produced by Bombyx mori from Those Produced by

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Discerning Silk Produced by Bombyx mori from Those Produced by Wild Species using ELISA Combined with Conventional Methods Qiushi You, Qingqing Li, Hailing Zheng, ZhiWen Hu, Yang Zhou, and Bing Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02789 • Publication Date (Web): 10 Aug 2017 Downloaded from http://pubs.acs.org on August 14, 2017

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Journal of Agricultural and Food Chemistry

Discerning Silk Produced by Bombyx mori from Those Produced by Wild Species using ELISA Combined with Conventional Methods Qiushi Youa, Qingqing Lia, Hailing Zhengb, Zhiwen Huc, Yang Zhoub,*, Bing Wanga,* a. Key Laboratory of Advanced Textile Materials and Manufacturing Technology, Ministry of Education, Zhejiang Sci-Tech University, Hangzhou 310018, China. b. Key Scientific Research Base of Textile Conservation, State Administration for Cultural Heritage, China National Silk Museum, Hangzhou 310002, China. c. Institute of Textile Conservation, Zhejiang Sci-Tech University, Hangzhou 310018, China * Email: [email protected] (B. Wang); [email protected] (Y. Zhou); Tel/Fax: +86-571-86843867

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Abstract: Recently, much interest has been attracted to separate silk produced by

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Bombyx mori from other species of silk and to trace the beginnings of silk cultivation

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from wild silk exploitation. In this paper, significant differences between silks from

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Bombyx mori and other species were found by microscopy and spectroscopy, such as

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morphology, secondary structure and amino acid composition. For further accurate

6

identification, a diagnostic antibody was designed by comparing the peptide

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sequences of silks produced by Bombyx mori and other species. The non-competitive

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indirect ELISA results indicated that the antibody which showed good sensitivity and

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high specificity candefinitely discern silk produced by Bombyx mori from wild

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species. Thus, the antibody-based immunoassay has the potential to be a powerful tool

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to trace the beginnings of silk cultivation. Besides, combining the sensitive, specific

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and convenient ELISA technology with other conventional methods can provide more

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in-depth and accurate information for species identification.

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Keywords: Bombyx mori, species identification, ELISA

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Introduction

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China is the birthplace of sericulture. As early as the Neolithic Age, the ancestors of

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the Chinese reeled off raw silk from wild cocoons, spun silk and wove fabrics. Later,

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a new industry of sericulture was started with the domestication of wild silkworms

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and wild mulberry trees. China’s high-quality silk is not only universally recognized

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as one of the most luxurious clothing materials but isalso an important part of the rich

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and ancient Chinese civilization. Therefore, studying the structure of silk in a range of

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species and distinguishing Bombyx mori silks from wild silks play an important role

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in tracing the beginnings of silk cultivation from the origins of wild silk exploitation

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in China.

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Chinese silkworms can be classified into Bombyx mori and wild silkworms,

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which are composed of many species, including the Eri silkworm, Antheraea pernyi,

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Philosamia cynthia Walker et Felder and the Millet silkworm. The silk cocoons and

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fibers produced by different silkworm species may have different morphological and

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structural characteristics1. It is necessary to study the morphologies and structures of

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silks and elucidate the differences as a basis for distinguishing them.

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Conventional methods, including scanning electron microscopy (SEM)2, 3,

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Fourier transform infrared spectroscopy (FTIR)4-6, X-ray diffraction (XRD)7, 8and

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amino acid analysis1, 9, among others, have been applied to study the longitudinal and

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cross-sectional features, characteristic peaks, secondary structures, and amino acid

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composition of silk fibers. However, the quality of cocoons and fibers is not only

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dependent on the species of the silkworms but is also affected by the rearing and 3

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spinning conditions1. Thus, a large number of diverse samples must be tested to

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establish a database consisting of morphological and structural features. In other

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words, different silkworm varieties may exhibit similar morphology and structure and

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conventional methods may have difficulty distinguishing them. Therefore, a new and

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reliable technology that can discern Bombyx mori from other species of silks should

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be established.

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The enzyme-linked immunosorbent assay (ELISA) has been widely used in

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biological research10-13 and is a promising diagnostic tool due to its advantages of

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good specificity, high sensitivity, easy operation, and low cost14, 15. Samples (antigens)

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interact with specific antibodies, which are conjugated with enzymes or fluoresceins.

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Then, the antigen-antibody conjugates combine with chromogenic or fluorogenic

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substrates and produce optical signals. The specific antibody is the most vital part of

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immunoassays; however, most of the specific antibodies used in tests are

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"off-the-shelf" and are not suitable for species identification. It is therefore necessary

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to design and prepare a tailored antibody for species identification. The fiber

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morphologies, secondary structures and chemical compositionsof different silks

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exhibit noticeable complexityand variability16-19, which can be traced to diversity

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inprotein amino acid sequences20. By comparing amino acid sequences of various

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silks, we found a diagnostic sequence and prepared a tailored anti silk fibroin (SF)

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antibody through peptide synthesis and carrier-protein coupling.

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Here, we combine conventional methods with ELISA to distinguish Bombyx

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mori from other species of silks. The morphologies, structures and amino acid 4

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compositions of diverse silks were determined by conventional methods, including

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universal microscopy (UM), SEM, ATR-FTIR, XRD and amino acid analysis. Then, a

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diagnostic antibody with the advantages of good specificity, easy operation and a low

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cost was prepared through peptide synthesis and carrier-protein coupling. Moreover,

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antibody-based ELISA was employed to identify the species of different silks. The

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results showed that combining the sensitive, specific and convenient ELISA

65

technology with other conventional methods can provide more in-depth and accurate

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information for species identification.

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Materials and Methods

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Reagents. Goat anti-rabbit IgG-HRP antibody (500 µg at 2 mg/mL), saline,

69

Freund’s complete adjuvant and Freund’s incomplete adjuvant were supplied

70

by

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sodium hydroxide, sodium carbonate, calcium chloride, sodium chloride,

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potassium

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monopotassium phosphate were supplied by Tianjin Gaojing Fine Chemical

74

Co., Ltd. Bovine serum albumin (BSA), human serum albumin (HSA),

75

ovalbumin, collagen I, sericin and keratin were purchased from Sigma-Aldrich.

76

Silkworm cocoons (Bombyx mori, Eri silkworm, Antheraea pernyi, Philosamia

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cynthia Walker et Felder, Millet silkworm) and silk fabrics (Bombyx mori, Eri

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silkworm and Antheraea pernyi) were provided by the China National Silk

79

Museum.

Hangzhou

Hua'an

chloride,

Biotechnology

disodium

Co.,

Ltd.

hydrogen

Hydrogen

peroxide,

phosphate

and

5

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Carbonate buffer (CB) solution at pH 9.6 was used as the diluent for

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ELISA antigens. PBS solution at pH 7.4 was used for wash steps. One percent

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BSA in PBS (pH 7.4) was used as the blocking solution for blocking unbound

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antigens and the diluent for the antibody. All other reagents were of analytical

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grade and used as received. The water used in all experiments was purified with

85

a TPM Ultrapure water system.

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Degumming. Cocoons and fabrics were degummed by treating twice in 0.5%

87

(w/w) Na2CO3 solution at 100 °C for 30 minutes witha bath ratio of 1:50. The

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insoluble silk fibers obtained after treatment were washed 3 times with water

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and dried at 60 °C overnight.

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Morphology. Images of the different species of cocoons were collected using a

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digital camera. The longitudinal features of the degummed silk fibers were observed

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using a scanning electron microscope (JEOL JSM-5610). Samples were sputtered

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with gold for 60 s at 15 mA and then measured at a typical accelerating voltage of 10

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kV. The fiber cross sections were obtained with a Type Y172 fiber slice cutter and

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examined using a universal measuring microscope (VANOX AHB-K1).

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Secondary structure. The infrared absorption spectra of degummed silks were

97

measured by attenuated total reflection Fourier transform infrared spectroscopy

98

(ATR-FTIR; Nicolet 5700, US) in the wavenumber range of 500–4000 cm-1. The

99

samples were directly analyzed with the spectrometer.

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X-ray diffraction measurements of degummed silks were carried out with a

101

Siemens D 5000 X-ray diffractometer with 40 Kv CU Ka and radiation of 30 mA. The 6

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silk fibers were powdered in a fiber slice cutter, and the powder was pressed to form a

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pellet. The pellet was fixed to the sample holder, and diffraction intensities were

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recorded from 5° to 50° (2θ) continuously ata scan rate of 3 °/min.

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Amino acid analysis. The amino acid contents and concentrations were analyzed via

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amino acid analysis (AAA; Waters 2695, America). To breakdown the proteins into

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the individual α-amino acids, the degummed silks were hydrolyzed with a 6 M

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hydrochloric acid solution. After hydrolysis for 24 h at 110 °C, the hydrated solution

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was dried with nitrogen. The hydrolysate and an internal standard substance were then

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dissolved in a derivation liquid and evaluated by AAA.

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Extraction of antigens. All the degummed silks and fabrics were extracted using

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the following method. One-tenth gram of shredded samples was added to the

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extracting solution (the molar ratio of calcium chloride-water-ethanol was 1:8:2)

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at a bath ratio of 1:50 and boiled for 3 h. The mixture was centrifuged for 10

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min at 8000 rpm. The supernatant (100µL) was transferred to a clean centrifuge

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tube, and 1 mL CB (pH 9.6) was added. The mixture was allowed to stand for

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10 min, followed by centrifugation for 10 min at 8000 rpm, to obtain the

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supernatant for coating.

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Silk fibroin powders of Bombyx mori used for the establishment of ELISA

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procedure were extracted as previously reported21. Briefly, silk was boiled

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twice in 0.5% (w/w) Na2CO3solution at a bath ratio of 1:100 for 0.5 h. Then,

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the insoluble silk fibroin was washed 3 times with water and dried at 60 °C

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overnight. Next, the shredded silk fibroin was added to the extracting solution 7

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(the molar ratio of calcium chloride-water-ethanol was 1:8:2) at a bath ratio of

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1:50 and boiled for 1 h. The solution was filtered to remove insoluble

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substances, and the obtained silk fibroin solution was dialyzed using a dialysis

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membrane with a molecular weight cut-off of 8000-10,000 for 3 days to

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remove calcium and chloride ions. Finally, the silk fibroin solutions were

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lyophilized to obtain silk fibroin powders.

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Preparation ofdiagnostic anti-SF primary antibody. The species-specific

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amino-acid sequence was determined based on the comparison of sequences of

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different silks. Based on the amino-acid sequences of the fibroin heavy chain of

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Bombyx mori, Eri silkworm, Antheraea pernyi, Philosamia cynthia Walker et

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Felder and Millet silkworm (Electronic Supplementary Material (ESM))

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retrieved

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(http://www.ncbi.nlm.nih.gov/pubmed/), the sequence MQRKNKNHGILGK of

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Bombyx mori was considered diagnostic and selected as the antigen for the

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silk-antibody preparation. The synthesis of the peptide MQRKNKNHGILGK

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was performed using a peptide synthesizer (CS Bio, California, USA). The

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immunogen

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keyhole-limpet hemocyanin (Sangon, Shanghai, China).

from

was

the

prepared

by

NCBI

coupling

public

the

synthetic

databases

peptide

with

142

The primary antibody was prepared as follows. First, 500 µg of

143

immunogen was diluted in saline and mixed with an equal volume of Freund’s

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complete adjuvant. Then, antibiotic solution was added to the mixture to form

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an emulsion. Ear blood samples of New Zealand white rabbits (14-16 weeks 8

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old) were drawn as controls before immunization. For the primary

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immunization, rabbits were injected subcutaneously with 100 mL of

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immunogen emulsions at multiple sites. Then, Freund’s complete adjuvant was

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replaced by Freund’s incomplete adjuvant to boost the immunization at 2, 4 and

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6 weeks after the primary immunization. Ten days after the third and fourth

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booster immunizations, the serum titer of the blood sample was measured by

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indirect ELISA. Sera were collected when the titer reached the required value.

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The resulting anti-SF antibody was further purified using a Protein A column

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and stored (3.15 mg/mL) at -20 °C for use.

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Indirect ELISA procedures. One hundred microliters of the extracted sample in CB

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(pH 9.6) was added to each well of amicrotiter plate and incubated at 4 °C overnight.

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After the solution was removed, the coated plate was washed 3 times with 200 µL

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PBS (pH 7.4). Next, 200 µL of BSA (1% in PBS 7.4) solution was added to the wells,

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and the plate was incubated at 37 °C for 1 h to reduce non-specific binding. The

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solution was removed, followed by 3 washes with PBS. Then, 100 µL of the diluted

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anti-SF primary antibody was added to the wells, followed by incubation at 37 °C for

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1 h. After the solution was removed, the wells were washed 3 times with PBS. Next,

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100 µL of goat anti-rabbit IgG-HRP antibody (secondary antibody) was added.

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After 1 h of incubation at 37 °C and a subsequent washing step, a 100 µL substrate

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solution (TMB color system) was added to the wells in a dark environment at room

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temperature for 10 min. Finally, 100 µL of 2 mol/L H2SO4 was added to terminate the

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color reaction and the sample absorbance was measured at λ=450 nm using a

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microplate reader (Model 550, Bio Rad).

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To ensure the effectiveness and specificity of the anti-SF primary antibody used

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in this experiment, several controls were employed. Silk fibroin of Bombyx mori was

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employed as a positive control, while PBS (pH 7.4) served as a negative control. For

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the blank control, anti-SF primary antibody was replaced by PBS (pH 7.4) solution.

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Five parallel wells were used for each sample and control and run simultaneously.

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The cut-off was defined as the mean OD450nm of CB (pH 9.6) plus three standard

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deviations (mean+3SD). Thus, OD450nm > cut-off was considered positive, while

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OD450nm ≤ cut-off was considered negative for the immunological test.

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All animal experiments were carried out in accordance with the national standard

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"Laboratory Animal-Requirements of Environment and Housing Facilities" (GB

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14925-2001) and the guidelines issued by the Ethical Committee of Zhejiang Sci-Tec

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University.

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Results

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Morphology. Silkworm cocoons had different shapes, sizes, color and morphological

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features, as shown in Fig. 1a-e. The cocoon of Bombyx mori, shown in Fig. 1a, had an

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oval shape, a white shiny appearance, and weighed approximately 400 mg. The Eri

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silkworm cocoon, shown in Fig. 1b, was slightly yellow in color and had an average

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weight of 411 mg. The cocoon had a fluffy outer layer, and fibers could be easily

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pulled from the cocoons before degumming. The Antheraea pernyi cocoon had a

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yellow color, as shown in Fig. 1c, and a fluffy appearance like the Eri silkworm 10

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cocoon. However, the Antheraea pernyi cocoon was heavier and had an average

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weight of approximately 874 mg. As shown in Fig. 1d, the Philosamia cynthia Walker

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et Felder cocoon had a conical shape and was brown in color, with a weight of 419

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mg. The Millet silkworm cocoon was unique among the cocoons studied in this

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research. The Millet silkworm cocoon was dark brown in color and had a net-like

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construction with a number of holes on the hard outer surface, as shown in Fig. 1e.

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The perforations in the cocoon allow air circulation to cool the pupa inside22, 23. The

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cocoon weighed approximately 391 mg and the fibers in the cocoons were tightly

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bound together by the gum.

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The longitudinal appearances of silk fibers from the five cocoons after

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degumming are shown in Fig. 1f-j. The Bombyx mori (Fig. 1f) fibers had a relatively

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smooth surface, whereas the Eri silkworm (Fig. 1g), Antheraea pernyi (Fig. 1h), the

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Philosamia cynthia Walker et Felder (Fig. 1i) and Millet silkworm (Fig. 1j) fibers had

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obvious striations, grooves or ridges. The Bombyx mori and Eri silkworm fibers

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appeared to be circular, whereas Antheraea pernyi, Philosamia cynthia Walker et

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Felder, and Millet silkwormfibers appeared to have a flat surface.

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Cross-sectional images of degummed silk fibers are shown in Fig. 1k-o. The

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Bombyx mori silks and wild silks exhibited different cross-sectional morphologies.

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The Bombyx mori silks had a triangular (Fig. 1k) cross section, where as the wild silks

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exhibited an elongated rectangular or a wedge-shape cross section. The average

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dimensions were listed in Table 1. The a/b (major axis/minor axis) values of the wild

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silks were much higher than those of Bombyx mori silks, indicating the flatness of 11

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wild silks. The wild silks also had a larger cross-sectional area, especially that of the

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Millet silkworm.

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Secondary structure. ATR-FTIR spectroscopy was used to investigate the molecular

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conformation of silks24-26. Fig. 2 depicts spectra gathered for different silks via

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ATR-FTIR. The Bombyx mori silks and all wild silks showed absorption peaks at

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1620 cm-1 and 1515 cm-1 in the amide I and II bands, which were attributed to β-sheet

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conformation. However, in the amide III band, the absorption peak at 1235 cm-1 was

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due to a random coil, while that at 1260 cm-1, which was assigned to β-sheet

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conformation, was more remarkable in silks produced by Bombyx mori. Furthermore,

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the most evident differencein the spectra was an absorption peak at 965 cm-1, which

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was characteristic of N–H rocking in “Ala-Ala” peptide structures and only existed in

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the wild silks. This peak was present due to the different sequence of amino acids

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used in the process of forming silk protein molecules. In wild silks, alanine and

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alanine are connected to each other to form the “Ala-Ala” peptide structure, while

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Bombyx mori silk proteins rarely form this structure27.

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All five spectra showed good resolution in the amide regions, making them

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suitable for qualitative analysis of protein secondary structures by deconvolution and

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curve fitting28. The amide III band was chosen to avoid the interference of

229

atmospheric water vapor. Fig. 3 showed the results of the peak recognition and

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deconvolution method. In the amide III band of Bombyx mori silk fibroin, the peak at

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1228 cm-1 was assigned to the random coil, helical conformation, or both, and the

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peak at 1260 cm-1 is assigned to β-sheet conformation in accordance with a previous 12

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study4. For the amide III band of wild silk fibroin, the 1215 cm-1 band was assigned to

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β-sheet conformation. Additional absorption bands at 1235 cm-1 and 1263 cm-1 were

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assigned to the random coil and α-helix conformations, respectively.

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The degree of crystallinity of silk fibers, which is closely correlated with the

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β-sheet content in silk, is usually determined by XRD29. Similar to ATR-FTIR, the

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diffraction peaks corresponding to the β-sheet conformation were different,

239

approximately 2θ = 20o for Bombyx mori silks and 2θ = 16o for the wild silks (Fig. 4).

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Table 2 compared the β-sheet content of Bombyx mori silks and wild silks obtained by

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ATR-FTIR and XRD. The values were obtained by calculating the areas of peaks that

242

were attributed to β-sheet conformations. The results for the β-sheet contents obtained

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by XRD were roughly in agreement with those by ATR-FTIR.

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Amino acid analysis. The composition of silk was determined in terms of its amino

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acid content. Table 3 provides the amino acid composition of the five silks. The

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amino acid content of Bombyx mori was considerably different from the wild silks. As

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shown in the table, the major amino acids, including alanine, glycine, serine and

248

tyrosine, accounted for 67 % of the amino acids in Millet silkworm fibers, whereas

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these four amino acids accountedfor approximately 80% in the other silks. The

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glycine content in Bombyx mori silks was higher than the alanine content, which was

251

the opposite of wild silks. Likewise, no histidine content was found in Bombyx mori

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silks, where as the amino acid level in wild silks was approximately 3%.

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Table 4 shows the ratios of amino acids in different species of silks. The wild

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silksshowed a higher ratio of basic to acidic amino acids: for instance, 0.99 for Eri 13

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silkworm, 0.80 for Antheraea pernyi, 0.61 for Millet silkworm, and 0.94 for

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Philosamia cynthia Walker et Felder, compared to 0.32 for Bombyx mori. Similarly,

257

the ratio of hydrophilicto hydrophobic components was high for all wild silks.

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Inaddition, the glycine/alanine ratio was very different for silkworm varieties. It was

259

approximately 3:2 for Bombyx mori silks and less than 1 for wild silks, averaging

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approximately 0.7. The amounts of glycine and alanine determine the crystallographic

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form of the proteins1, 30. The higher ratios of glycine/alanine in Bombyx mori silks

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suggest that the fibers may have good mechanical properties compared to wild silks31.

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Species identification of silks with ELISA

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Optimum antibody dilution. Optimal anti-SF antibody dilution, which reflected the

265

best specificity and sensitivity of the ELISA method, were obtained via parallel

266

titrations. To determine the optimum antibody dilution, serial dilutions of the primary

267

antibody were coated onto the wells followed by incubation with the secondary

268

antibody in gradient dilutions. The results are shown in Table 5. Under the same

269

dilution ratio for the anti-SF primary antibody, there was almost no difference in

270

optical density (OD) response for secondary antibody dilutions of up to 1:5000, but

271

asignificant reduction arose at the dilution of 1:8000. This finding indicates that there

272

were not enough secondary antibodies for the primary antibody to conjugate when the

273

dilution ratio for the secondary antibody was above 1:5000. Similarly, for each

274

secondary antibody dilution, an obvious reduction in OD response was again observed

275

when the anti-SF primary antibody was at a dilution of 1:2000, revealing that the

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coated sample could bind well with anti-SF primary antibody at a dilution of 1:1000. 14

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To avoid false-positive results and to maintain a low background, the lowest antibody

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concentrations giving a favorable OD450nm were chosen. Therefore, dilution ratios of

279

1:1000 for the anti-SF primary antibody and 1:5000 for the secondary antibody were

280

considered to be the optimal antibody dilutions. At these antibody concentrations, silk

281

fibroin showed a mean OD450nm value of 1.001, while PBS and BSA showed mean

282

OD450nm values of 0.185 and 0.164, respectively. In other words, the sensitivity of the

283

immunoassay was suitable for silk detection.

284

Sensitivity and specificity of anti-SF primary antibody. The ELISA protocol was

285

then employed to test the limit of detection (LOD) for fibroin. Silk fibroin was diluted

286

to a series of concentrations by CB (pH 9.6) and detected by indirect ELISA under

287

optimal antibody dilutions. The findings revealed that the LOD for silk fibroin was

288

approximately 100 ng/mL, indicating that silk fibroin cannot be detected using the

289

ELISA method when its concentration is below 100 ng/mL.

290

To enablea quantitative determination of the relationship between the OD

291

response and fibroin concentration, the OD value for standard fibroin solution was

292

plotted against the base-10 logarithm of the fibroin concentration. As shown in Fig. 5a,

293

the logarithm of the fibroin concentration and the OD value exhibitedan “S” type

294

trend. The logarithm of the concentration of fibroin and the OD value exhibited a

295

good linear relationship in silk fibroin concentrations ranging from 103 to 105 ng/mL

296

(OD values ranging from 0.438 to 1.01). This linear regression equation was as

297

follows:

298

y= 0.286logC-0.42 (R2=0.990)

(1) 15

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where C is the concentration of silk fibroin in ng/mL. The LOD of the assay,

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estimated as the concentration of the fibroin giving an OD value at 450 nm lower than

301

0.209 (cut-off), was 158.49 ng/mL.

302

The specificity of the antibodies was evaluated by cross-reactivity studies using

303

six possible interference antigens, including BSA, ovalbumin, collagen, HAS, keratin

304

and sericin. Samples were diluted with CB (pH 9.6) to obtain the same protein

305

concentration: 100 µg/mL. As shown in Fig. 5b, only the silk fibroin revealed a

306

positive result, while all other samples were negative, which illustrated that the

307

anti-SF primary antibodies used in the indirect ELISA do not show cross-reactivity

308

with any of the possible interference antigens tested. These results suggested that the

309

silk fibroin antibody is highly specific and can be broadly applied for the detection of

310

silk without interference from the possible interference proteins analyzed in this

311

study.

312

Identification of different species of silks. Once tested and optimized, the

313

developed method was used to identify silks from different species. The results

314

of silks are shown in Fig. 5c. Only the silks produced by Bombyx mori gave a

315

positive result, while all the wild silks showed negative results. Furthermore,

316

the OD values greatly differed between positive sample and negative samples,

317

which also indicated that ELISA has high specificity for discerning Bombyx

318

mori silk from wild species.

319

To further evaluate the effectiveness of the established ELISA method, three silk

320

fabrics produced by Bombyx mori, Eri silkworm and Antheraea pernyi were 16

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tested (silk fabrics produced by Philosamia cynthia Walker et Felder and Millet

322

silkworm couldn’t be found because they are seldom used for spinning process).

323

Fig. 6a shows the images of the fabrics. Silk fabric produced by Bombyx mori

324

had excellent performance properties such as smoothness, lightness, and

325

softness, while other fabrics appeared rougher, darker or harder. The ELISA

326

results of silk fabrics are shown in Fig. 6b. Silk fabrics produced by Bombyx

327

mori revealed an obvious positive result, while others samples behaved

328

negative, verifying the validity and practicability of the immunological method

329

in distinguishing Bombyx mori from other wild silks in actual fabrics.

330

Discussion

331

In our previous work, several anti-SF antibodies were designed and developed to

332

discern ancient silk from other textiles, such as cotton, hemp and wool32-34. However,

333

species identification of silk, which is crucial to trace the beginnings of silk

334

cultivation from wild silk exploitation, is still a key challenge. The reason for this

335

problem is mainly due to the similarity of homogenous proteins from different species,

336

varying degrees of fibroin degradation and the complexity caused by external

337

contamination. There are two main approaches to obtain the specific anti-SF

338

antibodies: one is to immunize rabbits with complete antigen (silk fibroin); the other

339

is constructing through immunizing animal with immunogen prepared by coupling the

340

hapten with carrier protein (keyhole-limpet hemocyanin). The haptens which

341

generally

342

GYGAGAGAGYGA) are synthesized using a peptide synthesizer. The antibodies

refer

to

diagnostic

sequences

(e.g.,

GAGAGSGAGAGS,

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343

obtained by both of the approaches show high sensitivity and specificity for the

344

identification of silk from other textiles. However, considering the sequence similarity

345

and structural homology of the fibroin from different species, only the latter approach

346

is suitable for discerning silk produced by Bombyx mori from those produced by wild

347

species.

348

Besides, significant differences between Bombyx mori silks and wild silks were

349

found by microscopy and spectroscopy, such as the longitudinal and cross-sectional

350

shape and size, characteristic absorption peaks, β-sheet content and amino acid

351

composition. However, conventional methods were challenged in distinguishing

352

different species of silks which have suffered from serious erosionand contamination.

353

For further accurate identification, a diagnostic antibody was designed by comparing

354

the peptide sequences of silks produced by Bombyx mori and other species. The

355

antibody showed good sensitivity and high specificity without cross-reactions with

356

other possible interfering antigens, indicating that the anti-SF primary antibody is an

357

acceptable antibody for detecting silk. The results also showed that ELISA can

358

definitely discern silk produced by Bombyx mori from wild species. Because ELISA

359

is especially suitable for the identification of poorly preserved ancient samples, we

360

may infer that the antibody-based immunoassay has the potential to be a powerful

361

toolto trace the beginnings of silk cultivation from wild silk exploitation. Besides,

362

combining the sensitive, specific and convenient ELISA technology with other

363

conventional methods can provide more in-depth and accurate information for species

364

identification. 18

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365

Abbreviations Used

366

SF, silk fibroin; LOD, limit of detection.

367

Acknowledgements

368

Financial support was provided by the National Natural ScienceFoundation of

369

China (51603188), the Public TechnologyResearch Plan of Zhejiang Province,

370

China under Grant No.2016C33175, and the Outstanding Young Research

371

Program of Science and Technology for the Protection of Cultural Relics

372

(No.2015-294).

373

Conflict of Interests

374

The authors declare no competing financial interests.

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Figure captions

Figure 1. Morphologies of different species of silks. First row, digital images of cocoons; second row, SEM images of fiber surface; third row, photomicrographs showing cross-sections of silk fibers. (a, f, k) Bombyx mori, (b, g, l) Eri silkworm, (c, h, m) Antheraea pernyi, (d, i, n) Philosamia cynthia Walker et Felder, and (e, j, o) Millet silkworm.

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Figure 2. ATR-FTIR spectra of different species of silks. B: Bombyx mori, E: Eri silkworm, A: Antheraea pernyi, P: Philosamia cynthia Walker et Felder, and M: Millet silkworm.

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Figure 3. Deconvolution results of amide III band of different species of silks. (a) Bombyx mori, (b) Eri silkworm, (c) Antheraea pernyi, (d) Philosamia cynthia Walker et Felder, and (e) Millet silkworm (circles, original spectrum; dashed curve, deconvoluted peaks; solid curve, simulated spectrum from summed peaks).

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Figure 4. X-ray diffractogram of different species of silks. (a) Bombyx mori, (b) Eri silkworm, (c) Antheraea pernyi, (d) Philosamia cynthia Walker et Felder, and (e) Millet silkworm.

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Figure 5. (a) Standard curves obtained at optimized antibody dilutions. (b) ELISA results for possible interference antigens. (c) ELISA results for different species of silks, B: Bombyx mori, E: Eri silkworm, A: Antheraea pernyi, P: Philosamia cynthia Walker et Felder, and M: Millet silkworm (The dashed lines refer to the cut-off value).

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Figure 6. (a) Digital images of different species of silk fabrics. (b) ELISA results for different species of silk fabrics. B: Bombyx mori, E: Eri silkworm and A: Antheraea pernyi (The dashed line refers to the cut-off value).

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Tables Table 1. Average cross-sectional dimensions of different species of silks.

Major axis(µm)

Cross-sectional

Minor axis(µm)

Variety

a/b area(µm2)

(a)

(b)

Bombyx mori

18.75

12.58

1.49

126.56

Eri silkworm

26.25

8.67

3.03

198.71

Antheraea pernyi

31.50

13.75

2.29

207.96

24.77

8.13

3.05

191.40

68.75

21.88

3.14

1201.67

Philosamia cynthia Walker et Felder Millet silkworm

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Table 2. Comparison of β-sheet content (%) of different species of silks obtained by ATR-FTIR and XRD Philosamia Bombyx

Eri

Antheraea

cynthia

Millet

mori

silkworm

pernyi

Walker et

silkworm

Method

Felder ATR-FTIR

21

20

20

38

30

XRD

56

47

45

53

56

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Table 3. Comparison of the amino acid composition of different species of silks. Philosamia Amino acid

Bombyx

Eri

Antheraea

Millet cynthia Walker et

composition

mori

silkworm

pernyi

silkworm Felder

Asp

2.85

6.15

8.76

5.69

8.48

Ser

13.20

7.66

12.14

7.52

12.64

Glu

2.39

1.48

1.87

1.41

2.98

Gly

35.55

26.42

21.74

27.33

16.65

His

0.00

3.82

2.90

3.34

3.14

Arg

1.16

3.30

5.25

2.95

3.40

Thr

1.31

0.59

0.58

0.64

0.79

Ala

24.47

34.69

32.20

35.26

25.83

Pro

0.73

0.73

0.70

0.71

0.85

Cys

0.05

0.00

0.00

0.04

0.00

Tyr

11.26

12.66

10.85

12.66

11.90

Val

3.21

0.65

1.05

0.63

4.19

Met

0.08

0.00

0.06

0.03

0.04

Lys

0.54

0.47

0.24

0.36

0.43

IIe

1.07

0.59

0.60

0.62

0.94

Leu

0.82

0.49

0.57

0.51

7.16

Phe

1.35

0.32

0.51

0.34

0.57

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Table 4. Comparison of the amino acid ratios of different species of silks Philosamia Amino acid

Bombyx

Eri

Antheraea

cynthia

Millet

composition

mori

silkworm

pernyi

Walker et

silkworm

Felder Basic/acidic

0.32

0.99

0.80

0.94

0.61

Hydrophilic/hydrophobic

0.49

0.57

0.74

0.53

0.78

Glycine/alanine

1.45

0.76

0.68

0.76

0.64

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Table 5. ELISA titration results for various controls treated with anti-SF primary antibody and secondary antibody. OD450nm±SD

Primary Antigen(concentration)

antibody (dilution, v/v)

Secondary antibody(dilution, v/v)

1:3000

1:5000

1:8000

silk fibroin (100µg/mL)

1:500

1.085±0.048 1.080±0.010 0.395±0.067

silk fibroin (100µg/mL)

1:1000

1.007±0.054 1.001±0.073 0.371±0.040

silk fibroin (100µg/mL)

1:2000

0.664±0.030 0.659±0.025 0.285±0.033

PBS

1:500

0.208±0.023 0.213±0.011 0.197±0.046

PBS

1:1000

0.171±0.016 0.185±0.010 0.155±0.036

PBS

1:2000

0.204±0.011 0.168±0.025 0.143±0.020

BSA (100µg/mL)

1:500

0.201±0.026 0.189±0.021 0.182±0.018

BSA (100µg/mL)

1:1000

0.198±0.034 0.164±0.033 0.172±0.054

BSA (100µg/mL)

1:2000

0.192±0.033 0.157±0.027 0.164±0.039

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