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

1

ACS Paragon Plus Environment


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

2

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

3

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

4

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

5

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

6

identification, a diagnostic antibody was designed by comparing the peptide

7

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

9

high specificity candefinitely discern silk produced by Bombyx mori from wild

10

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

11

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

12

and convenient ELISA technology with other conventional methods can provide more

13

in-depth and accurate information for species identification.

14

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,

18

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

19

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

20

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

21

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

22

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

23

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

24

in China.

25

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

26

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

27

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

28

fibers produced by different silkworm species may have different morphological and

29

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

30

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

31

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

32

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

33

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

34

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

35

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

36

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

40

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

42

be established.

43

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

45

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

46

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

47

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

48

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

49

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

50

"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

55

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

58

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

60

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

66

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

71

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),

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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

78

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

81

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

82

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

83

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

84

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

85

a TPM Ultrapure water system.

86

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

89

and dried at 60 °C overnight.

90

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

91

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

92

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

93

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

94

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

95

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

96

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.

100

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

103

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

104

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

105

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

106

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

107

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

108

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

109

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

110

dissolved in a derivation liquid and evaluated by AAA.

111

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

113

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

114

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

115

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

116

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

117

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

118

supernatant for coating.

119

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

121

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

122

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

123

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

125

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

126

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

127

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

128

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

129

lyophilized to obtain silk fibroin powders.

130

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

131

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

132

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

133

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

134

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

135

retrieved

136

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

137

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

138

silk-antibody preparation. The synthesis of the peptide MQRKNKNHGILGK

139

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

140

immunogen

141

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

144

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

145

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

147

immunization, rabbits were injected subcutaneously with 100 mL of

148

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

149

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

151

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

152

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

153

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

154

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

155

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

156

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

157

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

158

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

159

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

160

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

161

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

162

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

163

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

164

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

165

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

166

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

9



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

168

microplate reader (Model 550, Bio Rad).

169

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

170

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

171

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

172

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

175

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

176

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

177

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

179

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

180

University.

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Results

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

183

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

185

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

186

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

194

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

200

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

201

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

202

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

203

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

204

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

205

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

208

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.

213

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

215

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

216

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

217

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

218

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

219

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

220

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

221

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

222

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

223

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

224

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

225

Bombyx mori silk proteins rarely form this structure27.

226

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

227

suitable for qualitative analysis of protein secondary structures by deconvolution and

228

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

230

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

231

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

232

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

234

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

235

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

236

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

237

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

238

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).

240

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

241

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

243

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

244

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

245

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

246

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

247

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

249

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

250

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

252

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

254

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

256

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.

258

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

260

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

261

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

262

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

263

Species identification of silks with ELISA

264

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

276

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


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277

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

278

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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299

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

300

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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321

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,

17



Page 18 of 33

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


Page 19 of 33


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.

19



375

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21



Page 22 of 33

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.

22


Page 23 of 33


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.

23



Page 24 of 33

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).

24


Page 25 of 33


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.

25



Page 26 of 33

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).

26


Page 27 of 33


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).

27



Page 28 of 33

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

28


Page 29 of 33


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

29



Page 30 of 33

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

30


Page 31 of 33


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

31



Page 32 of 33

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


1:1000

1.007±0.054 1.001±0.073 0.371±0.040


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

32


Page 33 of 33


TOC Graphic

33


Discerning Silk Produced by Bombyx mori from Those Produced by

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