Research Article

Received: 20 September 2010 Revised: 18 December 2010 Accepted: 22 December 2010 Published online in Wiley Online Library: 18 February 2011

(wileyonlinelibrary.com) DOI 10.1002/jsfa.4321

Immunochromatography for the rapid

determination of cadmium concentrations
in wheat grain and eggplant
Kaoru Abe,a∗ Katsuo Nakamura,b Tomohito Arao,a Yasuhiro Sakurai,a
Ayumi Nakano,c Chieko Suginuma,d Kei Tawaradae and Kazuhiro Sasakif

Abstract
BACKGROUND: A simple and quick on-site test for trace levels of cadmium (Cd) in food is needed because of the human toxicity
of this heavy metal. We developed an immunochromatography kit which uses the antigen-antibody complex reaction between
the Cd–ethylenediaminetetraacetic acid (Cd–EDTA) complex and an anti-Cd–EDTA antibody. We previously reported the
successful use of this kit to determine Cd concentrations in brown rice with respect to the international standard: 0.4 mg kg−1 .
Here, we measured, using this immunochromatography kit, Cd concentrations in crops with lower international standards than
rice.

RESULTS: Cadmium extracted with 0.1 mol L−1 HCl from wheat grain and fresh eggplant was purified sufficiently using an
ion-exchange column treatment. Appropriate HCl extraction rates and dilution rates for the column eluate were selected; Cd
concentrations in wheat grain and fresh eggplant were determined successfully by immunochromatography with respect to
the international standards of 0.2 mg kg−1 and 0.05 mg kg−1 fresh weight, respectively.

CONCLUSION: Approximate Cd concentrations in wheat grain and fresh eggplant can be monitored easily and quickly by this
method at locations where facilities for acid digestion and precision analysis are not available.


c 2011 Society of Chemical Industry

Keywords: cadmium; anti-Cd–EDTA antibody; immunochromatography; wheat; eggplant; CODEX

INTRODUCTION and cost-effective.4,5 Recently, an immunochromatographic assay

Cadmium is one of the most toxic heavy metals for humans. More
than a few fields in Japan have been affected by Cd originating
from old mines and refining plants.1 Monitoring of cadmium in
agricultural products is needed to control cadmium in food.
Recently, the Codex Alimentarius Commission of the United
Nations Food and Agriculture Organization (FAO) and the World
Health Organization (WHO) adopted a new international standard
for Cd in foodstuffs.2,3 About 3.1% of 382 samples of wheat grain
(Triticum aestivum) and 5.8% of 400 samples of eggplant (Solanum
melongena) contained Cd concentrations above these limits (0.2
and 0.05 mg kg−1 , respectively) in a field and market basket study
in Japan.3 To prevent the distribution of polluted foods, a simple
and quick on-site test for Cd present at international standard
levels is needed.
However, measuring Cd concentrations in plants is costly and
troublesome, because it requires expensive analytical instruments
for such procedures as inductively coupled plasma optical
emission spectrometry (ICP-OES), inductively coupled plasma
mass spectrometry (ICP-MS), and flameless atomic absorption
spectrophotometry. These methods also require the services of
analytical experts and the use of exhaust systems that can remove
the toxic gases produced during acid digestion.
Immunochromatography is now widely used in medicine
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kit for detecting Cd in rice was developed by Kansai Electric
Power Co. of Japan.6,7 This method uses the antigen–antibody
complex reaction between the Cd–ethylenediaminetetraacetic
acid (Cd–EDTA) complex and an anti-Cd–EDTA antibody that
reacts specifically with this complex to detect Cd at concentrations
of 0.01 mg L−1 or higher; the results are read by the degree of
color developed on a test paper.6,7
∗
Correspondence to: Kaoru Abe, National Institute for Agro-Environmental
Sciences, 3-1-3 Kannondai, Tsukuba 305-8604, Japan.
E-mail: abekaoru@affrc.go.jp
a National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba
305-8604, Japan
b Sumika Chemical Analysis Service, Ltd, 3-1-135 Kasugade-naka, Konohana,
Osaka 554-0022, Japan
c Iwate Agricultural Research Center, 20-1 Narita, Kitakami 024-0003, Japan
d Saitama Prefectural Agriculture and Forestry Research Center, 1372 Kubojima,
Kumagaya, Saitama, 360-0831, Japan
e Kansai Electric Power Co., 1-7 Seikacho, Sourakugun, Kyoto 619-0237, Japan

f Central Research Institute of Electric Power Industry, 1646 Abiko, Abiko-shi,

for pregnancy and diagnostic tests because it is simple, fast Chiba 270-1194, Japan

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c 2011 Society of Chemical Industry
Immunochromatography to determine cadmium concentrations in wheat and eggplant
Figure 1. Process used to detect Cd in samples by using immunochromatography after extraction and then purification on an ion exchange column.
With this test, Cd in brown rice was extracted with HCl, and
the extract was purified on a chelate silicagel column prepared
based on Akatsuka et al.’s method8 to remove other metals. The
pretreated solution was then diluted with buffer for neutralization
and tested with the immunochromatographic assay.6 We have
already used this kit to determine Cd concentrations in various
brown rice samples with respect of an international standard:
0.4 mg kg−1 .9 – 11,12 The kit also has been useful for determining
Cd concentrations in rice foliage, soil and other crops.12,13 This
method does not require the services of analytical experts or the
use of expensive analytical equipment; the test can be performed
with apparatus commonly found in basic chemical laboratories.
A method is needed to simply and quickly measure Cd
concentrations in various kinds of crops with respect of their
international standards, but international standards of Cd for
various crops are lower than the standard for rice. In our previous
work, rice extracts were purified on a chelate silicagel column with
elution of Cd by nitric acid. For the subsequent immunoassay,
these samples had to be diluted with buffer by a factor of 20.
This process provided adequate sensitivity for rice, but insufficient
sensitivity for other crops with lower international standards. In
this study, to improve sensitivity we purified the extract on an
ion exchange column and eluted Cd with water. We measured,
by immunochromatography, Cd concentration in wheat grain
and fresh eggplant with respect to their international standards:
0.2 mg kg−1 and 0.05 mg kg−1 fresh weight (FW), respectively.
EXPERIMENTAL
The process used to measure Cd in samples by immunochromatog-
raphy is illustrated in Fig. 1. The method consists of an extraction
and purification process, and immunochromatography.
Extraction and purification
Wheat grain samples were produced in different fields in Japan and
eggplant samples were produced in fields and pots with polluted
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soil in Japan. Wheat grain was dried at 70 ◦ C at 72 h and milled
with a vibration cup mill (high-speed vibration sample mill TI-100,
CMT Co., Ltd, Iwaki, Japan). Cadmium in the samples was extracted
with shaking for 1 h in 0.1 mol L−1 HCl at a 1 : 10 grain : solution
ratio (w/v). Fresh eggplant was cut into small pieces and crushed
in a blender with distilled water of the same weight. For preparing
HCl extracts, 20 g juice was mixed well with 20 mL distilled water
and 10 mL of 0.5 mol L−1 HCl.
The wheat and eggplant extracts were filtered with filter paper
(qualitative no. 5B, Advantec Toyo, Tokyo, Japan), and 1 mL
of each filtrate was passed through an ion exchange column
(SEP-01, Sumika Chemical Analysis Service, Osaka, Japan), where
Cd was trapped selectively. This column was designed on the
basis of Akatsuka et al.’s method.8 It is believed that Cd forms
a chloro-complex and is trapped selectively by the column. The
column was then cleaned by adding 2 mL of 0.1 mol L−1 HCl to
remove coexisting compounds including other metals, because
Mn–EDTA, Zn–EDTA and Cu–EDTA each interfere with the
immunochromatography by cross-reacting to a small extent with
the anti-Cd–EDTA antibody.7 Cadmium retained in the column
was eluted with 1 mL distilled water.
Immunochromatography for Cd detection
Cadmium in the purified solution was detected with an im-
munochromatographic assay kit (Kadomieru immunochromato-
kit, SCAS, Osaka, Japan). The purified solution was mixed with
100 mmol L−1 Tris buffer solution containing 1.0 µmol L−1 EDTA
in order to neutralize the solution and produce Cd–EDTA (the anti-
gen in the immunochromatographic assay). The dilution rate with
the buffer was adjusted with respect to the international standard
for each crop. Forty microliters of the purified solution from wheat
grain was mixed with 360 µL EDTA buffer. Eighty microliters of
the purified solution from eggplant was mixed with 320 µL EDTA
buffer.
One hundred microliters of each sample solution was added to
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the freeze-dried, gold-colloid-labeled antibody, and the solution
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Figure 2. Concentrations of Cd and other metals in the solutions extracted from wheat grain and eggplant before and after purification. Average value
of three separate samples. Error bars represent standard deviations.
was then vortexed for 2–5 s. Under these conditions, the
Cd–EDTA complex binds to the anti-Cd–EDTA antibody to form
an antigen–antibody complex. The amount of this complex is
proportional to the amount of Cd in the original sample.
For the immunochromatography assay, 75 µL of the sample
solution was loaded onto the test apparatus, which consists of
nitrocellulose chromatography paper. A thin line of Cd–EDTA
antigen is fixed to the measurement line on the paper. When
the sample solution reaches the measurement line, antibody
not already linked to the antigen in the solution binds to the
antigen fixed to the line. The antigen–antibody complex is not
captured by the measurement line but passes through it. Antibody
captured on the line appears red because of the gold-colloid of
antibody. If large amounts of Cd are present in the measurement
sample, much of the antibody will have already bonded to the
antigen in the solution to form the antigen–antibody complex,
and only a small amount of unreacted antibody will be captured
on the measurement line, resulting in a light (red) color. In
contrast, if the sample contains little Cd, then a large amount
of antibody will be captured, resulting in a deep (red) color. After
40 min, the red band that appeared on the test line was read
with an immunochromatography reader (DiaScan 30-D, Otsuka
Electronics, Osaka, Japan). Three replicates were measured for
each sample.
Determining Cd concentration
Cadmium standard solutions (0.01 mol L−1 HCl) were measured by
immunochromatography. The dependence of the color readings
on Cd concentration follows a sigmoidal relationship, as is
usual for antigen–antibody reactions.9 – 13 A ‘concentration–color’
calibration curve was obtained by an exponential fit for Cd
concentrations in the range where a good linear relationship
was observed between the logarithm of Cd concentration and
the color readings. The equation of the curve was used to
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calculate the cadmium concentration of each sample solution
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K Abe et al.
(extracted–purified solution). Microsoft Excel was used for
calculation.
Chemical analysis
Wheat grain and cut eggplant were dried and milled. The Cd
concentrations of these samples were also measured by ICP-
MS (ICPM-8500, Shimadzu, Kyoto, Japan) after digestion with
concentrated HNO3 , followed by HF and HClO4 , in order to
evaluate the accuracy of the immunochromatographic assay. For
comparison, the Cd concentrations in the extracted solutions and
the extracted–purified solutions were also measured by ICP-OES
(Vista-Pro, Varian Inc., Palo Alto, CA, USA). We also measured Zn,
Mn and Cu in the extract solutions and the extracted–purified
solutions by ICP-OES in order to monitor the extraction and
purification processes.
RESULTS AND DISCUSSION
Purification of extract
In 0.1 mol L−1 HCl, not only Cd but also Zn, Mn and Cu were
extracted from the sample, and their concentrations were much
higher than the concentration of Cd (Fig. 2). Zn, Mn and Cu are
commonly present in natural samples, and their EDTA complexes
cross-react more with the Cd–EDTA antibody than with the
EDTA complexes of other metals.6 These excess coexisting metals
would compete with Cd for the antibody and thereby degrade
the accuracy of Cd detection by immunochromatography.6,9
Thus separation of other metals from Cd is necessary for the
accurate detection of Cd by the antibody. In our previous study,
a chelate silica gel column was successfully purified the Cd in HCl
extracts.7,11 – 13 Cadmium trapped by this chelate silica gel column
was eluted by 0.05–0.1 mol L−1 HNO3 solution. The column eluate
was diluted 20-fold with Tris buffer to neutralize it prior to
immunochromatographic assay. This method then successfully
determined [Cd] > 0.01 mg L−1 .7
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Immunochromatography to determine cadmium concentrations in wheat and eggplant www.soci.org

Figure 3. Relationship between color intensity and Cd concentration in the standard solution and the extracted–purified solutions of (a) rice (modified
from Abe et al.9 ); (b) wheat; and (c) eggplant. Vertical dark line: Codex maximum level for Cd.

To detect and quantify lower Cd concentrations, a lower buffer
dilution rate is necessary. In this study, we used an ion exchange
column from which trapped Cd can be eluted by distilled water in
order to decrease the dilution required to neutralize the column
eluate.
Large proportions of coexisting metals in the extracted solution
were removed from the extract by the column treatment (Fig. 2).
The ratios of the concentrations of Cu, Mn and Zn in the purified
solution to that of Cd were 15%, 26% and 140% for wheat grain,
and 5.9%, 1.3% and 3.8% for eggplant, respectively. However, the
cross-reactivities of Cu, Zn and Mn in the immunoassay were 1.4%,
0.72% and 0.57%, respectively.7 The reactivity of Cu, Zn and Mn in
the purified solution in the immunoassay was lower than 0.8% of
that of Cd. Therefore we considered the influence of these metals
on Cd detection during immunochromatography to be negligible
(Fig. 2). Cadmium in HCl extracts was purified successfully by the
ion exchange column.
Cd immunochromatography
In the Cd concentration range 0.01–0.1 mg L−1 in brown rice
extracts purified by chelate silica gel column with a rate of dilution
by buffer of 20 in our previous study,9 a good linear relationship
was observed between the logarithm of Cd concentration and the
color readings (Fig. 3(a)). As the rice was extracted with 0.1 mol L−1
HCl at a 1 : 10 brown rice : solution ratio (w/v), Cd concentration
in both extract and purified solution from rice containing the
international standard level of Cd (0.4 mg Cd kg−1 ) is 0.04 mg L−1 .
The rate of dilution by buffer was 10 for wheat grain – one half
a good linear relationship was observed between the logarithm of
Cd concentration and the color readings (Fig. 3(b)). Because Cd in
wheat grain was extracted with 0.1 mol L−1 HCl at a 1 : 10 wheat
grain : solution ratio (w/v), Cd concentration in both extract and
purified solution from wheat grain containing the international
standard level of Cd (0.2 mg Cd kg−1 ) is 0.02 mg L−1 . The rate
of dilution by buffer was 5 for eggplant – one quarter of that
for rice. In the Cd concentration range 0.0025–0.025 mg L−1 , a
good linear relationship was observed between the logarithm
of Cd concentration and the color readings (Fig. 3(c)). Because
Cd in eggplant was extracted with 0.1 mol L−1 HCl at a 1 : 4
eggplant : solution ratio (w/v), Cd concentration in both extract
and purified solution from eggplant containing the international
standard level of Cd (0.05 mg Cd kg−1 FW) is 0.01 mg L−1 . The
sensitivity for Cd quantification in eggplant was four times that in
rice (Fig. 3(a, c)).
Evaluation of Cd measurement by immunochromatography
The Cd concentrations in wheat grain determined by immunochro-
matographic assay were well correlated with the values obtained
by ICP-MS (r2 = 0.963; Fig. 4(a)). The slope of the linear regression
was 0.940 – quite close to 1. The Cd concentrations in eggplant
determined by immunochromatographic assay were also well cor-
related with the values obtained by ICP-MS (r2 = 0.949; Fig. 4(b)).
The average coefficient of variation (CV) was 15.5% for wheat grain
and 9.1% for eggplant. Abe et al.9 found an average CV of 14% for
brown rice. We therefore conclude that the approximate Cd con-
centrations in wheat grain and fresh eggplant can be determined
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of that for rice. In the Cd concentration range 0.005–0.05 mg L−1 , by immunochromatography.

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 www.soci.org K Abe et al.

Figure 4. Cd concentration in (a) wheat grain and (b) eggplant as measured by ICP-MS after acid digestion and immunochromatographic assay following
the extraction–purification process.

Table 1. Extraction and dilution rate for measuring food containing international standard level of Cd

HCl extraction Dilution with buffer

Cd in food (mg Food-to-HCl solution Approximate Cd in column eluate Column eluate-to-buffer

Food kg−1 ) ratio dilution rate (mg L−1 ) ratio Dilution rate

Rice 0.4 1 : 10 (w/v) 10 0.04 1 : 19 (v/v) 20

Wheat grain 0.2 1 : 10 (w/v) 10 0.02 1 : 9 (v/v) 10
Eggplant 0.05 1 : 4 (w/v) 5a 0.01 1 : 4 (v/v) 5
a Water content of eggplant is about 92%.

Table 1 summarizes HCl extraction rate and dilution rate of
column eluate to buffer in this study. By using the ion exchange
column for purification of Cd in HCl extract and diluting the column
eluate appropriately with buffer, we can measure Cd concentration
in food with respect to the international standard for ‘other
vegetables’: 0.05 mg kg−1 FW (Table 1). We concluded that we
can use this method to measure Cd concentration in crops with
respect to any level of Cd international standard.
We are now conducting an inter-laboratory study to validate
this method. Based on the inter-laboratory study results, we would
like to propose a threshold Cd concentration (cut-off value) for the
assay for the purpose of screening crops.
Approximate Cd concentrations in wheat grain and fresh
eggplant can be monitored easily and quickly by this method at
locations where facilities for acid digestion and precision analysis
are not available.
ACKNOWLEDGEMENTS
This work was supported in part by a grant from the Ministry of
Agriculture, Forestry and Fisheries of Japan (Research Project for
Ensuring Food Safety from Farm to Table AC-1261). We are grateful
to Ms Yukari Uchida, NIAES, for her assistance. We are also grateful
to Ms Yoko Hashimoto, NIAES, for chemical analysis.

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CONCLUSIONS
Cadmium extracted with 0.1 mol L−1 HCl from wheat grain and
fresh eggplant (at solution ratios of 1 : 10 and 1 : 4, respectively)
was purified sufficiently using an ion exchange column treat-
ment. When the column eluate of wheat grain was diluted with
buffer 10-fold, Cd concentrations more than 0.005 mg L−1 were
quantified by immunochromatography. When the column eluate
of eggplant was diluted with buffer fivefold, Cd concentrations
more than 0.0025 mg L−1 were quantified by immunochromatog-
raphy. Because appropriate HCl extraction rates were selected
and the column eluates were diluted at appropriate rates, Cd
concentrations in wheat grain and fresh eggplant were deter-
mined successfully by immunochromatography with respect to
the international standards: 0.2 mg kg−1 and 0.05 mg kg−1 FW,
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