Development of a highly sensitive and specific immunoassay for determining chrysoidine, a banned dye, in soybean milk film

Article abstract of DOI:10.3390/molecules16087043

A highly specific and sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA)was developed for the first time for the detection of chrysoidine, a dye banned in soybean milk film. Two haptens with different spacer arms were synthesized t

Full text of DOI:10.3390/molecules16087043

Molecules 2011, 16, 7043-7057; doi:10.3390/molecules16087043
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Development of a Highly Sensitive and Specific Immunoassay
for Determining Chrysoidine, A Banned Dye,
in Soybean Milk Film
Hongtao Lei 1, Jin Liu 1, Lijun Song 1, Yudong Shen 1, Simon A. Haughey 2, Haoxian Guo 1,
Jinyi Yang 1, Zhenlin Xu 1, Yueming Jiang 3,* and Yuanming Sun 1,*
1
Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural
University, Guangzhou 510642, Guangdong, China; E-Mails: hongtao@scau.edu.cn (H.L.);
liujin_001163@163.com (J.L.); songlijun0703330@163.com (L.S.);
shenyudong@scau.edu.cn (Y.S.); ghxghz@sina.com (H.G.); yjy361@163.com (J.Y.);
jallent@163.com (Z.X.)
2
Institute of Agri-Food and Land Use, Queen’s University Belfast, Belfast BT9 5AG, Northern Ireland,
UK; E-Mail: s.a.haughey@qub.ac.uk (S.A.H.)
3
Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical
Garden, Chinese Academy of Sciences, Guangzhou 510650, Guangdong, China
* Authors to whom correspondence should be addressed; E-Mails: ymjiang@scbg.ac.cn (Y.J.);
ymsun@scau.edu.cn (Y.S.); Tel.: +8620-37252525 (Y.J.); +8620-8528-3925 (Y.S.);
Fax: +8620-37252960 (Y.J.); Fax: +8620-85280270 (Y.S.).
Received: 18 July 2011; in revised form: 7 August 2011 / Accepted: 11 August 2011 /
Published: 17 August 2011
Abstract: A highly specific and sensitive indirect competitive enzyme-linked immunosorbent
assay (icELISA）was developed for the first time for the detection of chrysoidine, a dye
banned in soybean milk film. Two haptens with different spacer arms were synthesized to
produce antibodies. Both homologous and heterologous immunoassay formats were
compared to enhance the icELISA sensitivity. The heterologous icELISA exhibited better
performance, with an IC50 (50% inhibitory concentration) of 0.33 ng/mL, a limit of
detection (LOD, 10% inhibitory concentration) of 0.04 ng/mL, and a limit of quantitation
(LOQ, 20%–80% inhibitory concentration) from 0.09 to 4.9 ng/mL. The developed
icELISA was high sensitive and specific, and was applied to determine chrysoidine in
fortified soybean milk film samples. The results were in good agreement with that obtained
by high-performance liquid chromatography (HPLC) analyses.

Molecules 2011, 16
Keywords: chrysoidine; antibody; ELISA; soybean milk film
7044
1. Introduction
Chrysoidine (C.I. Basic Orange 2, Figure 1) is a type of industrial azoic dye [1]. Due to its good
dyeing fastness, it is widely used for dyeing leather, paper, feather, grass, wood, bamboo, etc. [2].
Chrysoidine can cause acute and chronic toxicity to mammals when taken by oral or skin route, or
inhaled, and its median lethal concentration (LC50, 24 h) for fish was 0.5 mg/L [3]. Chrysoidine has also
been recognized as a carcinogen [4] and its use in food has not been approved by any country.
Unfortunately, it has been reported that soybean milk film, a popular soybean food consumed in China
was adulterated with chrysoidine [5,6]. Moreover, chrysoidine has also been found in yellow-fin tuna
and dried bean curd stick [2,7]. Therefore, control of this banned dye in food is very crucial and the
development of a simple, economic, and rapid detection method is urgently needed.
Figure 1. Structure of chrysoidine.
The reported analyses of chrysoidine were mainly physio-chemical methods based on chromatography
with various detectors [2,4,5,8,9]. The detection limit of chrysoidin in a HPLC-MS study was
0.25 ng/g [4], and 2.3 ng/g in a GC-MS method [8]. Chromatography methods can provide accurate and
reliable results, but these methods are also expensive, laborious and time-consuming [10]. To date,
immunoassay technologies, especially ELISA, are increasingly replacing traditional chemical analyses
in screening of food contaminants and agrochemicals due to their sensitivity, time-efficiency and
cost-effectiveness [11-13]. ELISAs have been widely used for the determination of various
contaminants such as toxins [14,15], drugs [16,17], pesticides [18] and illegal additives [11,12] in the
biological, agriculture, and environmental fields. However, to the best of our knowledge, there is no
published literature on an immunoassay for the detection of chrysoidine.
In this study, a highly sensitive and specific ELISA for the determination of chrysoidine was
developed for the first time. Two chrysoidine haptens with different spacer arm lengths were synthesized
and covalently coupled to different carrier proteins to produce both immunogens and coating antigens.
The polyclonal antibody (pAb) to chrysoidine raised from immunized rabbits were characterized and
used for a competitive ELISA. The developed ELISA was further employed to analyze spiked soybean
milk film samples and validated by a HPLC method.

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2. Results and Discussion
2.1. Synthesis of Chrysoidine Haptens
For production of high quality antibodies and development of highly sensitive and specific
immunoassays, it is important to design a proper hapten structure. It was proposed that both the
conjugation position at the hapten molecule where the spacer is attached and the length of the spacer
may play an important role for a successful antibody production [11,19], and that the molecular
structure of the hapten should be left unchanged [20]. In this study two chrysoidine-derivatives with
different spacer lengths were synthesized (Scheme 1). One derivative with one carbon-atom spacer
length (Hapten 1) and the other with two-carbon-atom spacer length (Hapten 2) were modified at the
para position of the azo bond. The structures of Hapten 1 and Hapten 2 were confirmed by thin layer
chromatography (TLC), mass spectrometry (MS) and nuclear magnetic resonance (NMR) methods.
Scheme 1. Synthesis of chrysoidine haptens.
2.2. Synthesis of Immunogen and Coating Antigen
The chrysoidine derivative bearing a carboxylic acid group at the end of the spacer was activated by
the active ester method and then covalently coupled with a carrier protein (BSA or OVA) [21]. The
conjugates of hapten-BSA and hapten-OVA were used as immunogen and coating antigen, respectively.
Figure 2 shows the UV spectra of BSA, OVA, Hapten 1, Hapten 1-BSA and Hapten 1-OVA with
absorption peaks of Hapten 1, BSA and OVA at 450, 280 and 280 nm, respectively. The absorption
spectra of Hapten 1-BSA/OVA conjugates contain both absorption peaks of Hapten1 and BSA/OVA,
but with somewhat red shift. The results indicated that the coupling of hapten to BSA and OVA was
successful. Similar results were obtained with Hapten 2 conjugate (data not shown).

Molecules 2011, 16
Figure 2. UV spectra of BSA, OVA, Hapten 1, Hapten 1-BSA and Hapten 1-OVA.
7046
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Hapten 1-BSA
Hapten 1
BSA
OVA
Hapten 1-OVA
200
300
400
500
600
Wavelength(nm)
2.3. Optimization of icELISA Conditions
The prepared coating antigens were used for the icELISA assay. To improve the sensitivity of
icELISA, we have optimized the assay conditions by changing the assay format, concentration of the
coating antigen, dilution of the antiserum and antigen antibody reaction time. Two criteria were used for
optimizing the icELISA assay. (1) obtain a minimum IC50 value and (2) increase an absorbance to
0.8–1.5 units for the zero standard concentration (blank) [22].
The antisera collected after each boost were subject to titration and inhibition by the homology
indirect and competitive ELISA, respectively. All antisera showed relatively constant high titer and
inhibition ratio after the sixth immunization. Therefore, combinations of two different coating antigens
(Hapten 1-OVA and Hapten 2-OVA) and two antibodies (Ab 1 from Hapten 1 and Ab 2 from Hapten 2)
were tested. The experimental conditions, including coating antigen concentration and antiserum
dilution, were optimized for each combination (Table 1). In specific, coating antigen concentration and
antiserum varied in the range of 17–20 ng/mL and 1:4,000–1:20,000, respectively. The calculated IC50
values were found to be less than 11 ng/mL. With an IC50 of 0.33 ng/mL, the combination of hapten
1-OVA and Ab 2 was found to be most sensitive. With this combination the typical standard curve for
chrysoidine were constructed in the concentration range from 0.09 to 4.9 ng/mL and the values of LOD
at 10% inhibition was within 0.04 ng/mL, indicating that the sensitivity was better than that of the
reported physio-chemical method [4]. In addition, since both heterologous combination (Ab 1/hpaten
2-OVA and Ab 2/hapten 1-OVA) demonstrated lower IC50, significant differences in assay sensitivity
were observed between homogeneous and heterogeneous combinations of coating antigens and
antibodies (Figure 3), which implied that the heterologous plate coating antigens significantly
enhanced the assay sensitivity [12]. Thus, the combination of Ab 2 and hapten 1-OVA was used for the
further investigation due to its better sensitivity.

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Table 1. Optimized icELISA conditions.
Coating antigen
name
Coating concentration
Antibody
name
Ab 1
Antibody
dilution
8000
IC50
(ng/mL)
7.4
LOD
(ng/mL)
0.8
(ng/mL)
Hapten 1-OVA
Hapten 1-OVA
Hapten 2-OVA
Hapten 2-OVA
20
20
20
17
Ab 2
4000
0.33
0.04
Ab 1
20000
10000
1.6
0.4
Ab 2
10.6
3.8
Figure 3. icELISA standard curves for chrysoidine with heterogeneous combinations of
coating antigen and antibody. Data represented in mean ± SD (standard deviation) from
three assays. ■ combination of antibody 2 and Hapten 1-OVA; ● combination of antibody 1
and Hapten 2-OVA; ▲ combination of antibody 1 and Hapten 1-OVA; ▼ combination of
antibody 2 and Hapten 2-OVA.
100
80
60
40
20
0
10-4
10-3
10-2
10-1
100
101
102
103
104
chrysoidine (ng/mL)
2.4. Specificity of the icELISA
The assay specificity was evaluated using a set of compounds structurally related to chrysoidine,
and the cross-reactivity data for each compound is given in Table 2. The highest recognition was raised
from the two artificial haptens with CR values of 170%, 127%, respectively. The high CR to the
haptens could be explained by the similarity of the molecular structure [13]. The icELISA showed
lower cross-reactivity to Acid Yellow 23, orangeim and auramine O, the CR values were 0.04%, 0.12%
and 0.11%, respectively, which may be associated with the shared group benzene-ring, azo-bond
and amino group among these compounds. From Table 2 it is also apparent that there was no
cross-reactivity with other compounds, where CR values were less than 0.01%. As expected, the major
difference in the structures between chrysoidine and other dyes should be responsible for the low or no
recognition [11,23-25]. Although Hapten 1 and 2 showed more than 100% cross-reactivity, they both
are artificial and, therefore, have seldom chance to occur in food to cause the matrix effects. As a result,
the developed icELISA can meet the specificity requirement for the screening assay of chrysoidine.

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Table 2. Cross-reactivity of the polyclonal Ab 2 with chrysoidine, hapten and other
compounds based on coating antigen Hapten 1-OVA.
Compounds
Structure
IC50 (nmol/L) Cross-reactivity (%)
Chrysoidine
1.33
100.0
Hapten1
Hapten2
0.78
1.05
169.8
127.0
H2N
HOOC
N
N
NH2
Sunset yellow
a ND
≤0.01
Acid Yellow 23
Orangeim
3054.4
0.04
0.1
1121.90
Acid Red 94
ND
≤0.01
Auramine O
797.81
ND
0.2
1-(4-nitrophenyl-
azo)-2-naphthol
≤0.01
Sudan Red I
Azorubine
ND
ND
≤0.01
≤0.01

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Table 2. Cont.
Compounds
Structure
IC50 (nmol/L) Cross-reactivity (%)
Indigo
ND
ND
≤0.01
≤0.01
Allura Red
Quinoline
yellow
ND
ND
ND
≤0.01
≤0.01
≤0.01
Erythrosine
Tartrazine
Amaranth
ND
ND
≤0.01
≤0.01
OH
OH
O
O
HO
OH
OH
O
O
HO
HO
Carminic acid
Scarlet Base G
OH
OH
ND
ND
≤0.01
≤0.01
Reddish
orange
Carmine
ND
ND
≤0.01
≤0.01
Brilliant
blue-85

Molecules 2011, 16
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Table 2. Cont.
IC50 (nmol/L) Cross-reactivity (%)
Compounds
Structure
Orange yellow
ND
ND
ND
≤0.01
≤0.01
≤0.01
Gardenia
yellow
NH2
N
N
Melamine
H2N
N
NH2
a ND represents the infinite IC50 value that could not be fitted with the four-parameter logistic equation.
2.5. Fortification Experiment
The performance of an immunochemical method can be significantly influenced by the various
complicated matrices existing in the sample, and a dilution is usually a good way to eliminate this
matrix effect [26]. The effects caused by matrix were investigated using soya milk film purchased from
a local market (confirmed to be negative by HPLC). In this study, soybean milk film samples were
fortified with chrysoidine at a concentration of 45–180 ng/g and extracted with the extraction buffer
(0.1 M Na2HPO4:MeOH = 60:90, v:v). The absorbance of the 60-fold diluted sample extraction was
found to be almost same to that of the PBST (data not shown), indicating no obvious matrix effect.
Accordingly, the 60-fold dilution was selected for the subsequent experiments. The results obtained
from each spiked sample with five replicates are shown in Table 3. Recovery rates of 102.1%–106.5%
and intra-assay coefficients of variation of 2.4–4.9% were obtained.
Table 3. Recovery of chrysoidine from the fortified samples by icELISA.
Sample
weight (g)
2.5
Chrysoidine Fortified con. Dilution of Detected Recovery
CV (%)
(n = 5)
4.9
added (µg)
0.112
(ng/g)
45
extract
60
(ng/g)
48
(%)
106.5
102.1
103.1
2.5
2.5
0.225
90
60
92
3.4
2.4
0.450
180
60
186
2.6. Validation of icELISA by HPLC
The HPLC calibration curve for chrysoidine was constructed in the range of 0, 10, 20, 30, 90, 270,
810 and 2430 ng/mL. As indicated in Figure 4, chrysoidine can be detected at the retention time of
7.0 min using HPLC. The linear equation of the HPLC standard curve was Y = 122.4X − 359.0 (r2 = 0.99).
The detection and quantification limits, which were respectively defined as signal-to-noise ratio of 3:1
and 10:1, were 6.4 and 14.6 ng/mL, respectively.

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Figure 4. HPLC chromatogram of chrysoidine (10 ng/mL).
chrysoidine
To validate the applicability of the icELISA, the fortified food samples were measured by HPLC
and icELISA simultaneously. As shown in Figure 5, there is a good correlation between icELISA and
HPLC with the linear regression equation of Y = 1.03X – 70.79 (r2 = 0.99, n = 5). These results show
that icELISA can be useful for the primary quantitative screening of chrysoidine in soybean milk film.
Figure 5. Correlation of icELISA with HPLC analysis for soybean milk film samples.
1400
Y = 1.03 X - 70.79
r2 = 0.99
1200
1000
800
600
400
200
0
0
200
400
600
800
1000 1200 1400
ELISA (ng/g)
3. Experimental
3.1. Reagents
General reagents and organic solvents were of analytical grade unless specified. Chrysoidine (HPLC
grade), N,N’-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), 3,3’,5,5’-tetramethyl-
benzidine (TMB), bovine serum albumin (BSA), ovalbumin (OVA), 4-aminobenzoic acid,
4-aminophenylacetic acid, 3-aminobenzoic acid, m-phenylenediamine, complete and incomplete
Freund’s adjuvants were purchased from Sigma (St. Louis, MO, USA). HRP-conjugated

Molecules 2011, 16
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goat-anti-rabbit IgG was obtained from Boster Biotech Corporation Limited. (Wuhan, China).
Polystyrene ELISA plates were provided from Jincanhua Corporation (Shenzhen, China). Tween-20,
methanol (HPLC grade), dimethylformamide (DMF), dimethyl sulfoxide (DMSO) were obtained from
Yunhui Company (Guangzhou, China). All structure-related compounds such as Sunset yellow, Acid
Yellow 23, Orangeim, Acid Red 94, Auramine O, 1-(4-nitrophenylazo)-2-naphtho and Sudan Red I, etc.,
used for the cross-reactivity evaluation, were purchased from Dongzheng Chemical Corporation
(Guangzhou, China). Coating buffer: 0.05 M carbonate buffer, pH 9.8; Assay and washing buffer
(PBST): 0.01 M phosphate buffered saline with Tween-20, pH 7.4, containing 145 mM NaCl and 0.05%
(v:v) Tween-20; Blocking solution: 5% (v:v) skim milk powder in distilled water; Substrate solution:
15 mg TMB in 10 mL DMF, keep in dark place at 4 °C; Developing buffer: 0.04 M Na2HPO4·12H2O and
0.048 M citric acid; Stop solution: sulfuric acid (10%, v/v); Extraction buffer: 30 mL 0.1 M Na2HPO4,
150 mL H2O and 270 mL methanol to form 450 mL extraction buffer.
3.2. Instrumentation
UV-3010 spectrophotometer (Hitachi, Japan) was used for the UV-visible absorption measurements.
ELISA plates were washed with a microtiter plate washer DEM-3 (Tuopu, China). Absorbance was
measured at a wavelength of 450 nm using the Wallac 1420 VICTOR3 multilabel counter (Perkin Elmer
Ltd., US).
3.3. Hapten Synthesis
Two haptens used for immunizing and coating antigen are shown in Scheme 1. They were
synthesized by the same procedure using corresponding commercial start materials. The following
subsections describe the synthesis procedure and the identification of haptens.
3.3.1. 4-((2,4-Diaminophenyl)diazenyl)benzoic acid (Hapten 1)
To a stirring solution of 4-aminobenzoic acid (0.411 g, 3 mmol) in water (10 mL), cooled in an ice
water bath, 0.1 M hydrochloric acid (10 mL) was added to keep pH at 3–4, followed by dropwise
addition of sodium nitrite (0.228 g) dissolved in water (1 mL). The mixture was stirred for 2 h, and then
m-phenylenediamine (0.356 g, 3.3 mmol) dissolved in water (5 mL), pH adjusted to 6–7 with
hydro-chloric acid, and cooled with ice water was added dropwise. After stirring for 2 h, the mixture was
kept at 4 °C overnight and filtered to collect the residue. The crude product was purified by silica gel column
chromatography (chloroform-methanol = 15:1, v:v). Yield: 62%. TLC Rf =0.5 (CHCl3:MeOH = 15:1); MS
(APCI positive) m/z: 257 [M+H]+. 1H-NMR (DMSO-d6, 600 MHz) δ: 8.79 (s, 1H), 8.58 (s, 1H), 7.95 (d,
2H), 7.82 (d, 4H), 6.50 (d, 4H), 3.59 (d, 2H).
3.3.2. 2-(4-((2,4-Diaminophenyl)diazenyl)phenyl)acetic acid (Hapten 2)
Hapten 2 was synthesized using 2-(4-aminophenyl)acetic acid as the starting compound (Scheme 1).
The procedure is the same as the synthesis of Hapten 1 above. TLC Rf = 0.5 (CHCl3:MeOH = 15:1). MS
(APCI positive) m/z: 271 [M+H]+. 1H-NMR (DMSO-d6, 600 MHz) δ: 7.61 (d, 2H), 7.29–7.36 (d, 4H),
5.86–6.00 (d, 2H)

Molecules 2011, 16
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3.4. Preparation of Immunogen and Coating Antigen
The hapten was conjugated to BSA (bovine serum albumin; for use as immunogen) and OVA
(ovalbumin; for use as coating antigen) by the active ester method [12]. Briefly, to the each hapten
(0.1 mmol) in DMF (500 µL), DCC (0.2 mmol), followed by NHS (0.2 mmol) was added slowly. This
activation reaction was carried out overnight at 4 °C with continuous stirring. The reaction mixture was
centrifuged (10,000 g, 10 min) and, the supernatant was added very slowly to a BSA solution (150 mg
BSA or ovalbumin dissolved in 15 mL PBS). The mixture was stirred overnight at 4 °C to complete the
conjugation reaction. The mixture was then dialyzed against phosphate buffer (0.01 mol/L, pH 7.4) for
3 days (three changes of buffer per day). The immunogen and coating antigen were characterized by
the ultraviolet spectrophotometers [13]. The obtained immunogen hapten-BSA or coating antigen
hapten-OVA were stored at −20 °C until use.
3.5. Immunization Protocol
The intramuscular injection was given to one New Zealand white rabbits for each immunogen, total
two rabbits were used to raise antibody. In brief, each immunogen hapten-BSA (0.5 mg) was dissolved
in saline and emulsified with an equal volume of Freund’s complete adjuvant. Two New Zealand rabbits
weighing 1.5–2.0 kg were immunized four times using emulsified Hapten 1-BSA, Hapten 2-BSA,
respectively, at intervals of 21 days by the Guangdong Medical Laboratory Animal Center. The rabbits
were blood sampled to detect the presence of antibodies to chrysoidine using an indirect ELISA on the
eighth day after each immunization, starting 40 days after the first injection. Bleeding of animals was
performed 10 days after the fouth immunization and the collected antisera, named Ab 1 for that raised
from Hapten 1-OVA, Ab 2 for that raised from Hapten 2-OVA, respectively. Antisera were divided into
aliquots (1 mL) and stored at −20 °C until use.
3.6. Indirect Competitive ELISA
Checkerboard assays, in which antibodies were titrated against various amounts of the coating
antigen, were used to measure the reactivity of antibodies and to select an appropriate antigen coating
and antibody dilutions for competitive indirect assays. From the results of the checkerboard assays, two
antibodies were selected as the most suitable ones. Then, to select the most suitable coating antigen,
competitive assays were performed under various combinations at several chrysoidine concentration
levels. The concentrations of the antibodies and the coating antigen chosen were further optimized.
The procedure for the competitive assay was as follows. Microtiter plates were coated with
hapten-OVA (100–1000 ng/mL, 100 µL/well) in carbonate-bicarbonate buffer (50 mM, pH 9.6) by
overnight incubation at 4 °C. The plates were blocked with blocking solution by incubation for 3 h.
Serial dilution of the analyte in methanol-PBST (1:9, v:v, 50 µL/well) were added, followed by
50 µL/well of antibody diluted (1/1,000–1/10,000) with PBST solution. After incubation for about
30–60 min, 100 µL/well of a diluted (1/10,000) goat anti-rabbit IgG-HRP was added. The mixture was
incubated for 60 min, and 100 µL/well of a TMB solution was added. The reaction was stopped after an
appropriate time (typically 8 min) by adding 10% (v:v) H2SO4 (50 µL), and the absorbance was read at
450 nm.

Molecules 2011, 16
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3.7. Cross-Reactivity
Specificity of the optimized assay was tested by measuring the cross-reactivity using a group of
structurally related dyes. Twenty-four compounds were selected for testing the cross-reactivity (CR)
(Table 1). The CR was calculated as follows: CR(%) = [IC50(chrysoidine) / IC50(interferent)] × 100.
3.8. Sample Preparation
The soybean milk film samples were obtained from a local food supplier and were homogenized. To
the soybean milk film sample (2.5 g) chrysoidine standard (112.5, 225, 450 ng) was added in methanol
(100 µL). After setting aside for 1 h, extraction buffer (25 mL) was infused into the sample and the
extraction mixture was shaken for 20 min and supersonicated for 20 min, followed by centrifugation
(10,000 g) for 10 min. The supernatant was collected and diluted appropriately with PBST to be
measured by icELISA. For each sample, three separate extractions were performed and each sample was
determined in five replicates. Negative samples confirmed by HPLC were extracted in the same way and
used as a blank.
3.9. HPLC Analysis
Samples were extracted as described above just without the last PBST dilution step, after a filtration
with 0.45 µm membrane chrysoidine was analyzed on a Waters HPLC system equipped with a Waters
X-Brige-C18 column (4.6 × 150 mm, 5 µm). The injection volume was 10 µL. The mobile phase
consisted of methanol and 5 M ammonium acetate-acetic acid buffer (pH 4.17) at a volume ratio of
55:45. The flow rate was 1.0 mL/min. The detection wavelength was set at 458 nm. The retention time of
chrysoidine was 6.7 min. The concentrations of chrysoidine were calculated by calibration with the peak
areas of external chrysoidine standard.
4. Conclusions
In this work, antibodies against chrysoidine were prepared for the first time and used to develop an
indirect competitive ELISA. Two types of hapten have been synthesized and used to produce
polyclonal antibodies and coating antigens. Use of the antibody against the immunogen with
two-carbon-atom spacer arm and the heterologous coating antigen with one-carbon-atom spacer arm
resulted in the high sensitivity and specificity. The IC50 and LOD values of the assay for chrysoidine
are 0.33 and 0.04 ng/mL, respectively. All tested dyes exhibited no cross-reactivity with the antibody
to chrysoidine. In addition, high recovery rates (102.1%–106.5%) were obtained when this assay was
applied to the fortified samples. The ELISA method was also compared to HPLC with an excellent
correlation. Therefore, the icELISA could be a feasible quantitative/screening method for chrysoidine
analysis in food samples.
Acknowledgments
This work was supported by National Department Public Benefit Research Foundation
(201003008-08), SCAU Principal Fund on International Coorperation, China Guangdong Provincial

Molecules 2011, 16
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Science and Technology Projects (cgzhzd1005, cgzhzd0808, 2009B040500002) and the NSFC-RFBR
Joint Grant (21010206, 11-04-91189).
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Sample Availability: Samples of the compounds Sunset yellow, Acid Yellow 23, Orangeim, Acid Red
94, Auramine O, 1-(4-nitrophenylazo)-2-naphtho and Sudan Red I, etc., are available from the authors.
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