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Species identification of ancient leather objects by

the use of the enzyme-linked immunosorbent assay
Cite this: DOI: 10.1039/c6ay01816e
Yi Liu,a Yi Li,a Runxing Chang,a Hailing Zheng,b Yang Zhou,b Menglu Li,c Zhiwen Huc
and Bing Wang*a

Received 26th June 2016
Accepted 29th September 2016
DOI: 10.1039/c6ay01816e
www.rsc.org/methods
Leather is one of the indispensable necessities in human daily life. However, the identification of ancient
leather, especially species identification, is a great challenge for archaeologists and conservation
scientists. Fortunately, the non-competitive indirect enzyme-linked immunosorbent assay (ELISA) offers
a particularly promising approach for the analysis of ancient leather because of its advantageous
properties such as high efficiency, low-cost, and high sensitivity and specificity. This study focuses on
the use of a non-competitive indirect ELISA method to identify the species of ancient leather. Three
ancient leather samples, which were unearthed from the desert in the Xinjiang area, were characterized
using an analytical Oxford ISIS energy dispersive spectrometer (EDS), attenuated total reflection Fourier
transform infrared spectroscopy (ATR-FTIR) and ELISA. Two independent indirect ELISAs were
established, and the results from the two methods were used in the determination of animal species. It
was shown that all the three ancient leather samples were recognized by both anti-collagen-I antibody
(ab23446) and anti-collagen-I antibody (ab117119), which indicates that the species of these leathers
belong to cow. This is the first study to use an immunological method for the characterization of ancient
leather. It is concluded that the ELISA method has the potential to become a powerful analytical tool in
the identification of ancient proteinous materials.

The identication of artifacts made entirely or partially of

1 Introduction
Leather production is one of the most important and necessary
ancient industrial activities still in operation.1 Leather manu-
facture is an ancient art that was practiced by even the most
primitive races and practiced with very considerable skill by the
earliest of civilized peoples.2 In the ancient period, people
began to use animal skins to make a variety of leather products.
During the past several decades, there have been a number of
methods available for the identication of ancient leather, such
as Fourier transform infrared spectroscopy (FTIR),3–5 thermog-
ravimetric analysis (TGA),6–8 gas chromatography-mass spec-
troscopy (GC-MS),9,10 nuclear magnetic resonance (NMR)1,11–13
and electron paramagnetic resonance (EPR).1,14 However, most
of these methods are limited for accurate analysis and are not
suitable for the identication of ancient samples with
contamination or homogenous proteins from different species.
As a matter of fact, ancient leathers, which represent an
important part of the cultural patrimony, were usually buried in
tombs and subjected to deterioration and corrosion over time.15
a
Key Laboratory of Advanced Textile Materials and Manufacturing Technology,
Ministry of Education, Zhejiang Sci-Tech University, Hangzhou 310018, China
b
Key Scientic 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
leather is very challenging.16,17 This difficulty arises undoubt-
edly from the corrosion fragmented samples, complex combi-
nations of various ingredients and reduced availability of the
leather during the process of protein degradation.18,19 It is
a particularly difficult issue for archaeologists to investigate the
composition and origin of ancient leather, although the anal-
ysis of ancient leather has been an important eld of study for
a long time. Hence, the detailed characterization of ancient
leather is still a challenging issue in heritage science, and
therefore novel techniques should be introduced to obtain
insight into species identication in ancient leather.
In recent years, immunological techniques have attracted
increasing attention from professionals all over the world
involved in the study of cultural heritage.20–23 Immunological
techniques have the potential to become powerful diagnostic
tools in cultural heritage conservation for the highly specic
and sensitive identication of proteins in archeological mate-
rials and even microsamples of artifacts.24–26 Among the
immunological techniques, enzyme-linked immunosorbent
assay (ELISA) is popular and widely used in the scientic eld.27
The ELISA test offers several advantages over the traditional
methods used for ancient leather analysis, such as high sensi-
tivity and efficiency, relative specicity, and cost effectiveness.28–32
Furthermore, immunochemical methods are rapid, applicable to
routine analysis, have simple sample preparation and do not

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require expensive instrumentation.33 Therefore, the ELISA test
could make great contribution for the conservation of cultural
heritage.34
To date, there have been sporadic research about the appli-
cation of the ELISA method in cultural heritage, most of which
refer to paintings, binders, and proteinaceous material.33,35–38
For example, Cartechini et al. highlighted the most important
results achieved in the research on the development of analyt-
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ical methodologies based on the use of ELISA and immuno-
uorescence microscopy (IFM) techniques for the highly
sensitive and specic identication of proteins in artistic and
archeological materials.39 Palmieri et al. focused on an indirect
ELISA procedure for the specic identication of chicken-egg
yolk and animal glues in painting micro-samples.40 Hu et al.
combined the ELISA and IFM techniques to identify chicken
ovalbumin, glue from several mammalian species, bovine milk,
and sh glue in ancient Chinese painting samples.41 Zheng et al.
reported the preparation of a specic broin antibody and
successfully identied four silk samples from different time
periods.42 As a consequence, it is feasible to apply the ELISA test
in conservation science and systematically use it to develop
analytical methodologies.43
Our research work focuses on the species identication of
ancient leathers using a non-competitive indirect ELISA method
combined with other analytical methods. Briey, our analysis
includes three main steps. The rst part of this work describes
the characterization of ancient leather samples via EDS and
ATR-FTIR. The second part is about obtaining optimal dilutions
and evaluating the specicity of the anti-collagen-I antibody
(ab23446) and anti-collagen-I antibody (ab117119). The last and
most important part is the species identication of ancient
leather objects unearthed from the desert in the Xinjiang area.
The results reported herein provide a deep integration of bio-
analytical methods and the archaeological eld. The ELISA test
is a relatively new technique in conservation science, thus
providing a rich new eld for innovation, and it will play an
important role in cultural heritage research.
2 Experimental
2.1 Ancient samples
Three precious ancient leather samples were selected for
immunological identication. The samples were graciously
provided by the China National Silk Museum. All the samples
were excavated from the desert in the Xinjiang area, and were
dated in the period of 222-589 A.D., which was the Wei-Jin
Dynasties. The original condition of the samples is presented in
Fig. 1. S1 (Fig. 1A) was ancient leather from clothes in royal
tombs, S2 (Fig. 1B) was from gloves in royal tombs and S3
(Fig. 1C) was from shoots in royal tombs. All samples were in dry
condition and the items, which were unearthed in 19th century,
lost their exibility.
2.2 Reagents and chemicals
Mouse anti-collagen-I monoclonal antibody ab23446 (100 mL at
1 mg mL
1) and rabbit anti-collagen-I polyclonal antibody
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ab117119 (100 mL at 1 mg mL
1) were both purchased from
Abcam. Goat anti-mouse IgG (H + L) HRP conjugate (100 mL at
2 mg mL
1) and goat anti-rabbit IgG (H + L) HRP conjugate
(100 mL at 2 mg mL
1) were purchased from Hua'an Biological.
Collagen-I from bovine achilles tendon (1 g), urea, sodium
dodecyl sulfate (SDS), disodium ethylenediaminetetraacetate
dihydrate (EDTA), and albumin from bovine serum (BSA) were
purchased from Sigma-Aldrich. All other reagents were of
analytical grade and used as received. The water used in all
experiments was puried by a Milli-Q water system. Elution
buffer (EB) was prepared in advance: 180 g of urea was added to
a solution containing 5 mL of 1 mol L
1 Tris–HCl, 1 mL of
0.5 mol L
1 EDTA, and 25 mL of 20% SDS and diluted to
500 mL. The solution was adjusted to pH 7.4 and sterilized
before use. Phosphate-buffered saline solution (PBS) was
prepared as follows: 0.2 g KCl, 0.2 g KH2PO4, 8.0 g NaCl and
2.16 g NaH2PO4$7H2O were added to water and diluted to
1000 mL, and the solution was adjusted to pH 7.4 and sterilized
before use.
2.3 Energy dispersive spectrum (EDS) analysis
The element composition of the ancient samples was charac-
terized on an analytical Oxford ISIS energy dispersive spec-
trometer (EDS) system. The samples were sputter coated with
gold for 10 seconds and certain zones of the sample were
selected and detected at an accelerating voltage of 10 kV for EDS
analysis.
2.4 Attenuated total reection-Fourier transform infrared
(ATR-FTIR) spectroscopy
The infrared absorption spectra of the samples were measured
via the attenuated total reection Fourier transform infrared
spectroscopy (ATR-FTIR) technique (Nicolet 5700, America) in
the wavenumber range of 750–4000 cm
1.
2.5 Dissolution of collagen-I from bovine achilles tendon
The dissolution of collagen-I from bovine achilles tendon can
be summarized as follows. A solution containing 200 mL of 0.3%
(w/w) acetic acid was prepared in advance. Then, 20 mg pepsin
was completely dissolved in the solution. Meanwhile, 20 mg
collagen-I from bovine achilles tendon was also added in the
mixed solution. Next, the solution was incubated for 4 days at
a constant temperature of 37  C. Finally, the collagen-I from
bovine achilles tendon was completely dissolved and stored
(100 mg mL
1) at 4  C before use.
For the quantitative ELISA test, collagen-I from bovine
achilles tendon was diluted to 1, 2, 4, 6, 8, 10 and 12 mg mL
1
with EB solution and incubated at room temperature for 4 days.
The following procedures were the same as described above.
2.6 Pretreatment of samples
All samples (20 mg) were rst added to 2 mL EB solution and
incubated at room temperature for 4 days. Aer incubation, the
swelling of the ancient leather samples surface was observed,
which were broken down into many irregularly shaped
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Fig. 1 Digital images of the ancient leather samples unearthed from the desert in the Xinjiang area. (A) Ancient leather from clothes in royal
tombs (S1); (B) ancient leather from gloves in royal tombs (S2); and (C) ancient leather from shoots in royal tombs (S3). These pictures were taken
using a Canon EOS700D digital camera in the macro mode.
sediments subsided at the bottom of the container, while the
top was a clear solution. Finally, the supernatant liquid was
collected and used for the following ELISA test.
2.7 ELISA procedure
In this study, an indirect ELISA test was applied for species
identication in ancient leathers, in which the primary anti-
body is targeted by an enzyme-conjugated secondary antibody
instead of being conjugated with enzymes directly. The use of
a secondary antibody increases the specicity of ELISA and
reduces the unspecic background due to the linkage of anti-
bodies on the plastic surface of the well plate. In order to
investigate the specicity and sensitivity of the primary anti-
body used in this experiment, several negative and positive
controls were employed for the ELISA test. Elution buffer (EB)
solution and phosphate buffered saline (PBS) served as the
blank control and negative control, respectively, whereas
a solution of collagen-I from bovine achilles tendon served as
the positive control. Moreover, the optical density of the
samples at 450 nm was abbreviated as OD450 nm, and the dashed
line in each gure represents the detection line. The detection
limit was set as the mean OD450 nm value of PBS plus three times
the corresponding standard deviation.
First, 50 mL pretreated sample solution (10 mg mL
1) was
transferred to a micro-reaction vial and mixed with 100 mL of
100 mmol L
1 NaHCO3 solution and le to stand for 10 min.
Next, 15 mL of the mixed solution was added to each well of
a 96-well microplate (U-Bottom, 12w*8), followed by the addi-
tion of 65 mL 100 mmol L
1 NaHCO3 solution and incubation at
4  C overnight. Since the antigen being detected attaches to the
plastic of the well, the sample was removed and the plate was
washed with 300 mL PBS (0.01 M, pH 7.4) 3 times. Then 300 mL of
blocking buffer (1% BSA in PBS) was added and the plate was
covered again and incubated at 37  C for 1 hour to block the
non-specic binding sites in the coated wells. Aer washing
with 300 mL PBS 3 times, 80 mL of antigen-binding primary
antibody was added and incubated at 37  C for 2 hours. The
plate was then washed with 300 mL PBS (0.01 M, pH 7.4) 3 times
and the corresponding secondary antibody (used at an assay
dilution of 1/3000) was added to the wells and incubated at
37  C for 2 hours. Aer the removal of the secondary antibody
and washing with PBS 3 times, 100 mL of substrate solution
(TMB color system) was added to each well in a dark environ-
ment at room temperature for 10 minutes. Finally, 100 mL of
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1 mol L
1 H2SO4 was added to terminate the color reaction and
the sample absorbance at l ¼ 450 nm was measured using
a microplate reader (Model 550, Bio Rad).
Obtaining optimal antibody dilutions that correspond to the
best specicity and sensitivity plays an important role in the
ELISA method. It is also the key factor in the ELISA test to
identify the ancient leathers. In the ELISA test, PBS served as the
negative control, whereas collagen-I from bovine achilles
tendon served as the examined antigen, which is the positive
control. In order to get the best dilution ratio of each primary
antibody, high sensitivity and low negative value should give
a good compromise in the antibody optimization. In this study,
both anti-collagen-I antibody (ab23446) and anti-collagen I
antibody (ab117119) were diluted by 1% BSA solution to
different concentrations, and then the primary antibody was
targeted by the corresponding goat anti-mouse IgG (H + L) HRP
conjugated and goat anti-rabbit IgG (H + L) HRP conjugated
secondary antibody, respectively. Finally, the mean optical
density (OD) values were measured on a microplate reader
(Model 550, Bio Rad). Moreover, the values of “ODCol/ODPBS”
were employed as criteria to evaluate the appropriate primary
antibody concentration. To retain high sensitivity, primary
antibody concentrations with a high OD450 nm positive control
(collagen-I) value was chosen for the detection of the ancient
leather samples. Meanwhile, a low negative value should not be
ignored. The larger the ODCol/ODPBS value, the greater the
difference between the OD450 nm values of collagen and PBS.
The negative value was relatively low, accordingly.
3 Results and discussion
3.1 EDS analysis of the ancient samples
For preliminary characterization, the basic elements of the
ancient leather samples are intuitively presented by EDS anal-
ysis. As shown in Fig. 2A, the selected and detected zone of S1
was sprinkled with large amounts of irregularly shaped sedi-
ment which adhered onto surfaces. The coating is composed of
C, O, N, Al, Na, Mg, S, K, and Ca elements, and the atomic
fraction is C ¼ 49.57%, O ¼ 37.53% and N ¼ 8.79%, which
account for the vast majority of the total elements. Also, in
Fig. 2B, the morphology of S2 is shown as a tough surface with
many irregular solid objects. The coating is composed of C, O,
N, and Al elements without other impurities, and the atomic
fraction is C ¼ 48.54%, O ¼ 44.91% and N ¼ 4.95%, which still
occupy most of the element composition. As shown in Fig. 2C,
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Fig. 2 EDS spectrograms of the ancient leather samples, S1 (A); S1 (B) and S3 (C). The zones marked by the red rectangle were selected and
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detected for the analysis of the samples.
the surface of S3 is much smoother than the other two samples,
with many pieces of aky crystals on it. The coating is composed
of C, O, N, Al, Na, Mg, S, Cl, and Ca elements, and the atomic
fraction is C ¼ 46.96%, O ¼ 26.5% and N ¼ 17.32%. The content
of N element was much higher than that of S1 and S2. In
conclusion, all the three ancient leather samples show sharp
and intense peaks of C, O and N, which indicate the main and
indispensable composition of collagen. The Al, Na, Mg, S, K, Ca
and Cl elements were from the desert area soil of the tombs.
EDS analysis was only able to determine the existence of
elements, and cannot determine specic chemical composi-
tion, thus FTIR was further carried out in the following analysis.
3.2 ATR-FTIR analysis of the ancient leather samples
ATR-FTIR is widely used to characterize the internal structure
and chemical composition of ancient relics. As shown in Fig. 3,
all three samples show one peak at 3309 cm
1, which is assigned
to the stretching vibration of N–H in the protein backbone. Also,
the sample spectra show the characteristic peaks of amide
bonds, specically a sharp peak at 1645–1642 cm
1 (amide I
band), a peak at 1542–1548 cm
1 (amide II band) and a broad
shoulder at 1010–1040 cm
1 (amide III band). Besides, the peak
at 1452 cm
1 belongs to the stretching vibration of C–C bonds,
whereas the weak absorption peak in the range of 1239 cm
1 to
1408 cm
1 is the bending vibration of C–H in protein. Above all,
aer burial in the desert in the Xinjiang area for a long time, the
basic components and the integrity of the protein molecule
Fig. 3 ATR-FTIR spectra of the ancient leather samples.
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structure of the ancient leather samples were maintained and
could be successfully detected. However, it is still necessary to
implement a highly sensitive and specic method, i.e. ELISA, for
the accurate species identication of the ancient leathers.
3.3 Optimization of experimental parameters
As shown in Fig. 4A, with an increase in the dilution of the
primary antibody, the OD450 nm values of collagen-I decreased
rst and then increased, whereas the OD450 nm values of PBS
uctuated to some extent. By comparison, when the dilution
ratios of anti-collagen-I antibody (ab23446) were 1 : 200 and
1 : 1000, the OD450 nm values of collagen-I were larger than those
at other concentrations. Accurately, the values of “ODCol/ODPBS”
under the dilution ratios of 1 : 20 000, 1 : 10 000, 1 : 8000,
1 : 5000, 1 : 3000, 1 : 1000 and 1 : 200 were 2.21, 2.60, 1.98, 1.67,
2.31, 3.45 and 2.22, respectively. Especially, under the dilution
ratio of 1 : 1000, the value of “ODCol/ODPBS” was the largest and
the standard deviation was much smaller than the others.
Therefore, the dilution ratio of the anti-collagen-I antibody
(ab23446) was set as 1 : 1000 for the following experiment. As
shown in Fig. 4B, the values of “ODCol/ODPBS” under the dilu-
tion ratios of 1 : 10 000, 1 : 8000, 1 : 5000, 1 : 3000, 1 : 1000 and
1 : 200 were 3.60, 3.46, 4.55, 4.24, 4.07 and 4.88, respectively.
Although the value of “ODCol/ODPBS” at the dilution ratio of
1 : 200 was larger than that of the dilution ratio of 1 : 5000, the
higher the concentration of the primary antibody, the greater
the probability of obtaining false positive results, which is not
conducive to archaeological research. Moreover, in view of the
antibody titer and production costs, the dilution ratio of anti-
collagen-I antibody (ab117119) was set as 1 : 5000. At this point,
there was a good compromise between the high positive value
and low negative value, which ensures the high efficiency and
sensitivity of the ELISA test for the identication ancient
leather. To sum up, the optimal dilution multiple of the anti-
collagen-I antibody (ab23446) and anti-collagen-I antibody
(ab117119) was 1 : 1000 and 1 : 5000, respectively.
3.4 The specicity of primary antibodies
According to the instruction for the anti-collagen-I antibody
ab23446, this monoclonal antibody is highly specic for type I
collagen. The native triple-helical conformation is required for
reaction. It has no cross reactivity with type II, III, V and VI
collagens. There is no evidence for cross reactivity with other
connective tissue proteins (laminin, bronectin, and elastin).
Also, it can react with sheep, goat, cow, dog, human and pig,
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Fig. 4 ELISA titration results for different conditions. (A) The anti-collagen-I antibody (ab23446) dilution ratio is 1 : 20 000, 1 : 10 000, 1 : 8000,
1 : 5000, 1 : 3000, 1 : 1000 and 1 : 200; and (B) the anti-collagen I antibody (ab117119) dilution ratio is 1 : 10 000, 1 : 8000, 1 : 5000, 1 : 3000,
1 : 1000 and 1 : 200. The concentration of collagen-I is 10 mg mL
1. 50 mL of collagen-I solution was used for each ELISA test. Goat anti-mouse
IgG (H + L) HRP conjugate and goat anti-rabbit IgG (H + L) HRP conjugate were used as the corresponding secondary antibodies for ab23446 and
ab117119, respectively. The dilution ratio of the two secondary antibodies is 1 : 3000. Each dot of the figure represents the mean  SD (standard
deviation) of n ¼ 5 assays.

whereas it cannot react with mouse, rat, rabbit, horse, chicken,
guinea pig, cat and kangaroo. The other anti-collagen-I anti-
body, ab117119, is specic for bovine collagen type I and cross
reacts weakly with human collagen type I. Negligible reactivity
has been observed with chicken collagen I, bovine collagen II, IX
and XI and bovine bronectin.
Further investigation was applied in the next ELISA test to
evaluate the specicity of anti-collagen-I antibody (ab23446)
and anti-collagen-I antibody (ab117119). The ELISA procedure
for the recognition of collagen-I began with the antibody panel
titration of standard solutions. The specicity of the primary
antibody was tested by checking cross-reactivity with a series of
possible interference antigens, including BSA, silk broin, and
keratin. As shown in Fig. 5A, the OD450 nm values of the inter-
ference antigens, i.e. BSA, silk broin and keratin, were similar
with the blank and negative controls, and all their values were
below the dashed line. On the contrary, the OD450 nm value of
collagen-I was especially high, and above the detection line,
which clearly showed a positive result. Equally, as shown in
Fig. 5B, all the OD450 nm values of the possible interference
antigens were less than 0.2 except collagen-I. Moreover, the
OD450 nm values of the interference antigens were below the
dashed line, which show a negative result, while the OD450 nm
value of collagen-I was particularly high, even close to 0.85,
which shows an obvious positive result. The above results
proved the effectiveness and specicity of the anti-collagen-I
antibody (ab23446) and anti-collagen-I antibody (ab117119).
There was no cross-reactivity with a series of possible interfer-
ence antigens. This further indicates that the two primary
antibodies are acceptable for the species identication of
ancient leathers.
3.5 Species identication of ancient leather objects
Since the ELISA procedure was optimized, it was ultimately used
for the species identication of ancient leather objects

Fig. 5 ELISA results for various possible interference factors treated with two types of primary antibodies. (A) Interference antigens were reacted
with anti-collagen-I antibody (ab23446); and (B) interference antigens were reacted with anti-collagen I antibody (ab117119). The concentrations
of all the proteins are 10 mg mL
1 50 mL of protein solution was used for each ELISA test. Goat anti-mouse IgG (H + L) HRP conjugate and goat
anti-rabbit IgG (H + L) HRP conjugate were used as the corresponding secondary antibodies for ab23446 and ab117119, respectively. The dilution
ratio of the two secondary antibodies is 1 : 3000. Each column of the figure represents the mean  SD (standard deviation) of n ¼ 5 assays. The
dashed line shows the test criterion (the mean ODPBS plus three times the corresponding standard deviation). If the OD450 nm is above the dashed
line, the result is positive, and vice versa.

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Fig. 6 ELISA results for the ancient leather samples. (A) Anti-collagen-I antibody (ab23446) was treated with the ancient leather samples; and (B)
anti-collagen I antibody (ab117119) was treated with the ancient leather samples. Each column of the figure represents the mean  SD (standard
deviation) of n ¼ 5 assays. The dashed line shows the test criterion (the mean ODPBS plus three times the corresponding standard deviation). If the
OD450 nm is above the dashed line, the result is positive, and vice versa.
unearthed from the desert in the Xinjiang area. As shown in
Fig. 6A, S1, S2, S3 and collagen-I all showed positive results,
while EB (blank control) and PBS (negative control) gave nega-
tive results. Given that the anti-collagen-I antibody (ab23446) is
highly specic for type I collagen and it can react with sheep,
goat, cow, dog, human and pig, this indicates that the animal
species of these ancient leather samples were from one or more
of these animals. Furthermore, as shown in Fig. 6B, S1, S2, S3
and collagen-I all tested positive to anti-collagen-I antibody
(ab117119) and their OD450 nm values were much higher than
that of the EB and PBS controls. Since the ab117119 antibody is
specic for bovine collagen type I and cross reacts weakly with
human collagen type I, sheep, goat, dog and pig are ruled out as
the species origin of these ancient leathers. Moreover, the most
commonly used animal skin to make leather were those of
domesticated cattle, sheep, goat, and to a lesser extent pig.44,45
To date, to the best of our knowledge, there is no literature that
mentions leather materials originating from humans. It is
illegal and irrational to use human skin for leather production,
Fig. 7 ELISA calibration curves obtained for collagen-I from bovine
achilles tendon, expressed as the relationship between OD450 nm and
the collagen-I concentrations.
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both in the past and present. Thus, it is concluded that the
animal species origin of these leathers was actually cow.
In order to estimate the content of collagen-I in the ancient
samples, a quantitative ELISA test using a standard curve for
collagen-I was further performed. As shown in Fig. 7, the rela-
tionship between OD450 nm and the collagen-I concentrations is
described in the ELISA calibration curves, and the data reveals
a good linear relationship. With the OD450 nm values of ancient
leather samples under the dilution ratio of 1 : 5000 for anti-
collagen-I antibody (ab117119), the concentration of collagen-I
in S1, S2 and S3 can be calculated according to the standard
curve. The calculated concentration of collagen-I in S1, S2 and
S3 was 8.074 mg mL
1, 6.254 mg mL
1 and 4.475 mg mL
1
respectively. Next, in combination with the sample concentra-
tion (10 mg mL
1), the content of collagen-I in S1, S2 and S3 was
calculated to be 80.74%, 62.54%, and 44.75%, respectively. This
indicates that there were signicant differences in the residual
collagen-I content among the ancient leather samples. Briey,
a novel method for the species identication of ancient leather
has been successfully introduced, which may play an important
role in archeology and heritage conservation.
4 Conclusions
This study focused on the selective species identication of
ancient leathers. It is an example of the valuable outcomes that
can be attained from interdisciplinary approaches based on
analytical and bioanalytical methods. Combining the rapid,
sensitive and specic ELISA technique with other complemen-
tary routine analytical methods can provide more information
regarding ancient leathers. As far as we know, this is the rst
study to use an immunological method for the characterization
of ancient leathers. Furthermore, the results provide a deeper
insight into the interactions between antibodies and immuno-
reactive antigens in ancient samples, and a novel method for
the species identication of ancient leathers has been
successfully introduced. Briey, immunological techniques
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have the potential to become a particularly useful tool at
archaeological sites and conservation science laboratories.
Acknowledgements
Financial support was provided by the outstanding young
research program of science and technology for the protection
of cultural relics (2015-294), Special Funds from the Adminis-
Published on 30 September 2016. Downloaded by Vanderbilt University Library on 13/10/2016 15:26:11.
tration of Cultural Heritage of Zhejiang Province (2014) and
Science Foundation of Zhejiang Sci-Tec University (ZSTU) under
Grant No. 13012141-Y.
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