Development of an enzyme-linked immuno-sorbent assay (ELISA) method for carbofuran residues

Article abstract of DOI:10.3390/molecules13040871

The haptens 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)carbonyl]- amino]butanoic acid (BFNB) and 6-[((2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)- carbonylamino]hexanoic acid (BFNH) were synthesized and then used to develop a rapid, specific and sensitive ELISA method to determine residues of the pesticide carbofuran in a variety of matrices. A hybridoma cell line (5D3) producing anti-carbofuran monoclonal antibodies (MAbs) was also established. Based on the MAbs in combination with the heterologous hapten BFNH coupled to either horseradish peroxidase (HRP) or ovalbumin (OVA), four ELISAs (formats I-IV) for the quantification of carbofuran were developed and compared. Among them, the optimized format II (the conjugate-coated direct competitive ELISA) showed the best characteristics, with an IC₅₀ value of 18.49 ng/mL, a limit of detection of 0.11 ng/mL and the shortest assay time (1 h). This ELISA method was then applied to the determinations of carbofuran in environmental water, soil and food samples. The relative standard deviations (R.S.D.s) ranged from 1.8% to 21.3% and the mean recoveries were 104.6%, 108.3%, 106.3% and 100.1% for water, soil, lettuce and cabbage, respectively. Thus, the ELISA method of format II exhibited the potential to develop commercial ELISA kits for a rapid detection of carbofuran for human health and environmental safety.

Full text of DOI:10.3390/molecules13040871

Molecules 2008, 13, 871-881
molecules
ISSN 1420-3049
2008 by MDPI
www.mdpi.org/molecules
©
Full Paper
Development of an Enzyme-linked Immuno-Sorbent Assay
ELISA) Method for Carbofuran Residues
(
1
1
2
1,*
1
1
Jinyi Yang , Hong Wang , Yueming Jiang , Yuanming Sun , Ke Pan , Hongtao Lei , Qing
1
1
1
1
Wu , Yudong Shen , Zhili Xiao and Zhenlin Xu
1
College of Food Science, South China Agricultural University, Guangzhou 510642, P. R. China; E-
mails: yjy361@163.com; gzwhongd@163.com; xiaopan412@163.com; hongtao@scau.edu.cn;
wuqingq@263.net; shenyudong@scau.edu.cn; scau_xzl@163.com; jallent@163.com
South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, P. R. China; E-
mail: ymjiang@scbg.ac.cn
2
*
Author to whom correspondence should be addressed; E-mail: ymsun@scau.edu.cn
Received: 27 March 2008; in revised form: 14 April 2008 / Accepted: 14 April 2008 / Published: 17
April 2008
Abstract: The haptens 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)carbonyl]-
amino]butanoic acid (BFNB) and 6-[((2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)-
carbonylamino]hexanoic acid (BFNH) were synthesized and then used to develop a rapid,
specific and sensitive ELISA method to determine residues of the pesticide carbofuran in a
variety of matrices. A hybridoma cell line (5D3) producing anti-carbofuran monoclonal
antibodies (MAbs) was also established. Based on the MAbs in combination with the
heterologous hapten BFNH coupled to either horseradish peroxidase (HRP) or ovalbumin
(OVA), four ELISAs (formats I-IV) for the quantification of carbofuran were developed
and compared. Among them, the optimized format II (the conjugate-coated direct
competitive ELISA) showed the best characteristics, with an IC50 value of 18.49 ng/mL, a
limit of detection of 0.11 ng/mL and the shortest assay time (1 h). This ELISA method was
then applied to the determinations of carbofuran in environmental water, soil and food
samples. The relative standard deviations (R.S.D.s) ranged from 1.8% to 21.3% and the
mean recoveries were 104.6%, 108.3%, 106.3% and 100.1% for water, soil, lettuce and
cabbage, respectively. Thus, the ELISA method of format II exhibited the potential to
develop commercial ELISA kits for a rapid detection of carbofuran for human health and
environmental safety.

Molecules 2008, 3
872
Keywords: Carbofuran, monoclonal antibodies (MAbs), enzyme-linked immuno-sorbent
assay (ELISA)
Introduction
Carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranylmethyl carbamate) is a pesticide widely and
effectively used to control insects, but it is a potent cholinesterase inhibitor and thus exhibits a high
toxicity to human beings and wildlife. Because of its widespread use carbofuran residues are potential
air, soil, water and food pollutants [1,2]. In recent years, some assays such as gas-chromatography
(
[
GC), electro-chromatography, high-performance liquid chromatography (HPLC) and immunoassay
3,4] have been developed to detect carbofuran residues present in water or foods. Among these
assays, the HPLC method with post-column derivatization and fluorescence detection is currently the
preferred one [5,6], but the method requires specialized instrumentation and is time-consuming.
Recently, attention has been paid to the enzyme-linked immunosorbent assay (ELISA) as an
analytical technique in the agrochemical field as it provides analytical chemists with a rapid and
sensitive method [7-9]. Liu et al. [10] and Zhu et al. [11] have reported an immunoassay method to
detect carbofuran residues, but the antibodies used were polyclonal, which had the deficiencies of
limited specificity, affinity and purity. Abad et al. [12-14] developed an ELISA method for detecting
carbofuran residues based on monoclonals and applied it to analyze fruit and vegetable samples, but
they only used one or two ELISAs, and the detection limit for carbofuran was only 10 ng/mL, a
because of this relatively high detection limit it was unsuitable as an analytical method for developing
a commercial ELISA kit for the sensitive detection of carbofuran residues. In this study, special
monoclonal antibodies (MAbs) were produced and four ELISAs for detecting carbofuran residue were
developed. The best ELISA was selected by comparing the four different formats and it was then
applied to determine carbofuran residues in water, soil, lettuce and cabbage samples.
Results and Discussion
Antigen synthesis
The haptens 6-[((2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)carbonylamino]hexanoic acid
(
BFNH) and 4-[((2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)carbonylamino]butanoic acid (BFNB)
1
were synthesized by a literature method [13] (Scheme 1). The products were characterized by their H-
NMR spectra, which matched the reported data [13].
These haptens were then covalently attached to bovine serum albumin (BSA) using the modified
active ester method [15]. The UV-vis spectra showed the qualitative differences between the carrier
protein and conjugate in the maximum absorbance regions of the haptens.

Molecules 2008, 3
873
Scheme 1. Synthesis of the carbofuran haptens BFNB and BFNH.
The hapten to protein molar ratio can be estimated from the spectral data. As the molar absorptivity
of haptens was the same for the free and conjugated forms, the apparent molar ratios were estimated as
2
0 and 13 for the haptens BFNH and BFNB, respectively. After evaluation of the additive absorbance
values, the molar ratios of haptens to protein were evaluated as 5 and 3 for BFNH and BFNB,
respectively, based on the covalently attachment of the haptens to ovalbumin (OVA) [16].
Preparation and purification of MAbs
A hybridoma cell line (5D3) producing anti-carbofuran MAbs was established. The titres of culture
3
6
medium and ascites were up to 1:2.048 × 10 and 1:1.024 × 10 , respectively. The MAbs were then
purified by saline precipitation with saturated ammonium sulfate. The concentration of obtained MAbs
was 1.135 mg/mL.
Preparation of antibody-HRP
The MAbs were covalently attached to HRP using the periodate oxidation method [17] and then the
conjugate was determined by UV spectrophotometry. The OD403nm and OD280nm values were
determined to be 0.297 and 0.658, respectively. The molar ratio of HRP to antibody (E/P) was 1.35.
The E/P was reported to be suitable for further used when it was between 1 and 2 [17].
Evaluation of four ELISA formats
On the basis of the use of the MAbs together with BFNH-OVA, BFNH-HRP and Antibody-HRP
conjugates, four immunoassay formats (Format I-IV) were developed. The four ELISAs were
established as a result of an optimization process, which included a study of the concentration of
MAbs and the conjugates, the temperature and time. The inhibition curves of four ELISAs to
carbofuran are shown in Figure 1 and their characteristics were summarized in Table 1. The most
prominent findings of this study were the BFNH-OVA coated format ELISAs (format I and format II)
produced a highly sensitive assay. The analytical characteristics of the immunoassays listed in Table 1
were estimated from the standard curves depicted in Figure 1. The limit of detection, estimated as the
analyte concentration giving a 10% inhibition, and the assay working range, calculated as the analyte

Molecules 2008, 3
874
concentrations providing a 20-80% inhibition, were very different for the four ELISA formats. This
fact is clearly shown in Table 1. Among them, format I exhibited the best sensitivity and limit of
detection, but it required more steps and took longer A favorable ELISA format requires both good
sensitivity and limit of detection, and should involve less steps and be time saving [18,19], so on this
basis the format II was determined to be the best one, with an IC value, a limit of detection and
5
0
duration being 18.49 ng/mL, 0.11 ng/mL, and 1 h, respectively. It was deemed suitable for preparation
of the commercial ELISA kits for detection of carbofuran residues.
Figure 1. Carbofuran inhibition curves using the four test formats.
100
Format I
Format II
1
00
8
6
4
2
0
0
0
0
8
6
4
2
0
0
0
0
0
0
1
E-4 1E-3 0.01 0.1
1
10 100 1000 1000 01 00000
1E-4 1E-3 0.01 0.1
1
10
100 1000 10000
[CARBOFURAN] (ng/mL)
[CARBOFURAN] (ng/mL)
6
0
Format III
Format IV
1
8
6
4
2
0
8
6
4
50
1
1
1
1
40
30
20
10
0
1
E-4 1E-3 0.01 0.1
1
10 100 1000 1000 01 00000
1E-4 1E-3 0.01 0.1
1
10 100 1000 10000
[CARBOFURAN] (ng/mL)
[CARBOFURAN] (ng/mL)
Differences between the four ELISA formats
The study showed that formats I and II were better than formats III and IV because the number of
the HRP-hapten conjugates recognized by immobilized antibodies was lower than the number of
OVA-hapten conjugates recognized [20,21]. Moreover, hapten with a large spacer arm and with short
linking groups exhibited less suitability for enzyme conjugates, probably due to steric hindrances [22-
24].

Molecules 2008, 3
875
Table 1. A summary of the four carbofuran immunoassays.
Format
Parameter
Format I
.5
Format II
0.25
Format III
—
Format IV
—
BFNH-OVA
ng/mL)
0
(
MAbs (ng/mL)
0.057
Peroxidase-
labeled goat anti-
mouse
—
0.35
1.42
1 μg/mL
The secondary
antibody
—
—
Goat anti-mouse
immunoglobulins
immunoglobulins
diluted 1:20000
—
Antibody-HRP
BFNH-HRP
1:80
—
3
—
1:800
3
—
1:400
4
—
5
Step
Assay time
4
1
1.5
2
(
h)
IC (ng/mL)
11.63
18.49
6343.38
>10000
5
0
Limit detection
10% inhibition)
ng/mL)
(
0.007
0.11
0.066
3.58
(
Linearity
IC -IC
IC -IC
IC -IC
IC -IC
15
2
0
80
20
80
20
50
8
detection range
0
.08-1694.93
0.65-692.64
9.82-6343.38
0.74-188.92
(
ng/mL)
—
Not applicable
Sample analysis
Water, soil, lettuce and cabbage samples were spiked with carbofuran at 0.1, 1, 10 and 50 ng/mL
and directly analyzed by ELISA (Format II). The results were summarized in Table 2. The mean
recoveries for water, soil, lettuce and cabbage juices were 104.6%, 108.3%, 106.3% and 100.1%,
respectively. The R.S.D.s were ranging from 1.8% to 21.3%. The recoveries and precisions obtained
for the four samples can be considered good for a residue detected method. In this study, the samples
without carbofuran were used as the negative control.
Conclusions
In this study a conjugate-coated direct competitive ELISA (format II) for detecting carbofuran, with
an IC50 value of 18.49 ng/mL and a limit of detection of 0.11 ng/mL, was developed. The ELISA was
applied to determine carbofuran residues in water, soil, lettuce and cabbage samples. The R.S.D.s
ranged from 1.8% to 21.3% and the mean recoveries were 104.6%, 108.3%, 106.3% and 100.1% for
water, soil, lettuce and cabbage, respectively. Thus, the ELISA method of format II exhibited the
potential to develop commercial ELISA kits for a sensitive and rapid detection of carbofuran residue.

Molecules 2008, 3
876
Table 2. Analyses of carbofuran in water, soil, lettuce and cabbage samples by ELISA (n = 3).
Fortification
Mean±S.D.
Recovery
Matrix
R.S.D. (%)
Level (ng/mL)
( ng/mL)
0.14±0.03
0.93±0.04
9.71±0.11
47.6±0.93
(%)
140.0
93.0
97.1
95.2
106.3
120.0
87.0
98.0
95.2
0
.1
14.7
12.8
5.9
1
Lettuce
10
5
0
12.2
11.4
13.9
13.7
6.4
Means
.1
0
0.12±0.02
0.87±0.06
9.80±0.25
47.6±1.02
1
1
0
0
Cabbage
Water
5
21.3
Means
.1
100.1
130.0
96.0
13.8
7.7
0
0.13±0.01
0.96±0.078
9.42±0.17
49.18±4.35
1
8.1
10
94.2
1.8
5
0
98.4
8.8
Means
.1
104.6
130.0
107.0
98.8
6.6
0
0.13±0.02
1.07±0.11
9.88±0.88
48.69±5.72
15.6
10.4
8.9
1
Soil
10
5
0
97.4
11.7
11.6
Means
108.3
Experimental
Materials
Analytical pesticide standards were obtained from Shennong Jiangsu Chemistry Co. The BSA and
OVA were purchased from Sigma-Aldrich. Enzyme immunoassay grade HRP, peroxidase labelled
rabbit anti-mouse immunoglobulins and rabbit anti-mouse immunoglobulins were obtained from
Wuhan Boster Biotech Co. All other chemicals and organic solvents were of reagent grade.
Polystyrene ELISA plates were from Guangzhou Jiete Biotech Co. and treated with a Multiskan MK2
microplate washer (Thermo Labsystems). Absorbances were read with a Multiskan MK3 microplate
reader (Thermo Labsystems). Ultraviolet-visible (UV-vis) spectra were recorded on a UV-160A
1
Shimadzu spectrophotometer (Kyoto, Japan). H-NMR spectra were obtained on a DRX-400 NMR
spectrometer (Bruker, Germany-Switzerland).
Antigen synthesis
Hapten synthesis [13]. 2,3-Dihydro-2,2-dimethyl-7-benzofuranol was reacted with a slight excess of
phosgene at 25°C for 4 h to give 2,3-dihydro-2,2-dimethyl-7-benzofuranyl chloroformate (DDBC),

Molecules 2008, 3
877
which was subsequently used without further purification. After the crude DDBC (7.14 g, 31.4 mmol)
reacted for 1.5 h with aminocaproic acid (4.60 g, 35.1 mmol) or aminobutyic acid (3.62 g, 35.1 mmol),
the precipitated product was collected, then washed with water, and finally dried to yield crude 6-
[
((2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)carbonylamino]hexanoic acid (BFNH, 7.8 g, 66%
yield) or 4-[((2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)carbonylamino] butanoic acid (BFNB, 5.6
g, 52% yield), which were recrystallized from hexane/ethyl acetate (70:30) to yield the pure haptens,
1
whose identities were confirmed by their NMR spectra. BFNH: H-NMR: δ 1.42 (s, 6H, 2CH ),
3
1
.41−1.63 (m, 6H, CH
2
CH
2
CH
2
), 2.25−2.29 (t, 2H, CH
2
COOH), 3.02 (s, 2H, CH
2
), 3.15−3.20 (q, 2H,
1
CH NH) and 6.70−6.96 (m, 3H, aromatic)]; BFNB: H-NMR: δ 1.40 (s, 6H, 2CH ), 1.83 (m, 2H, CH ),
2
3
2
2 2 2
2.39 (t, 2H, CH COOH), 3.01 (s, 2H, CH ), 3.23 (q, 2H, CH NH) and 6.69−6.96 (m, 3H, aromatic)].
These results were a good match the data reported by Abad et al. [13], confirming the successful
synthesis of both compounds.
Preparation of immunizing conjugates. The haptens BFNB and BFNH were covalently attached to
BSA by the modified active ester method [15] and the resulting conjugates were determined by UV
spectrophotometry. The molar ratio of hapten to protein was calculated by the method of Chen et al.
[16].
Preparation of coating conjugates. The haptens BFNB and BFNH were covalently attached to OVA
using the mixed-anhydride method [15], and were determined as mentioned above.
Production of MAbs to carbofuran
BALB/c female mice (8−10 weeks old) were immunized with BFNB-BSA conjugates. The
splenocytes of immunized mice were fused with Sp2/0 cells using PEG 1500 as the fusing agent. The
culture supernatant of hybridoma cells was screened to determine the presence of antibodies that
recognize carbofuran. The screening consisted of the simultaneous performance of noncompetitive and
competitive indirect ELISAs, to test the ability of antibodies to bind the OVA conjugate of the
immunizing hapten and to recognize carbofuran, respectively. The selected hybridomas were cloned
by the micrurgical technique. Stable antibody-producing clones were expanded and cryopreserved in
liquid nitrogen.
Purification of MAbs
The hybridomas were collected after centrifugation and then suspended in the culture media without
fetal bovine serum. The BALB/c female mice received a soluble intraperitoneal injection at a dose of
6
6
0
.5 mL and a cell concentration of 1×10 /mL to 2 × 10 /mL. The ascites were abstracted after two
weeks. The antibodies were purified by saline precipitation with 45% saturated ammonium sulfate and
the concentration of antibodies was then determined by spectrophotometry.

Molecules 2008, 3
878
Preparation of antibody-HRP
The MAbs were covalently attached to HRP using the periodate oxidation method [17]. The HRP
5 mg) was dissolved in distilled water (1 mL) and sodium periodate (0.1 mol/L) was then added. The
(
reaction was incubated at 25°C for 20 min. The product was dialysed at 4°C for 12 h with sodium
acetate buffer (1 mmol/L) at pH 4.0. The antibody was diluted to 10 mg/mL with sodium carbonate
buffer (pH 9.5, 0.1 mol/L). The pH value of the actived HRP was then adjusted to 9.5 with sodium
carbonate buffer (pH 9.5, 0.1 mol/L). The antibody (0.5 mL) was added to the HRP solution and the
mixture was then allowed to react at 25°C for 2 h. Sodium borate (4 mg/mL) was added and reacted at
4
°C for 2 h. Finally, the product was dialysed with 0.1 mol/L sodium phosphate buffer (pH 7.4). The
conjugate was determined by UV spectrophotometry. The molar ratio of HRP to antibody (E/P) was
calculated as follows:
HRP (mg/mL) = OD403 nm × 0.4
IgG (mg/mL) = (OD280 nm- OD403 nm × 0.3) × 0.62
E/P = (HRP × 4) / IgG
(1)
(2)
(3)
Preparation of BFNH-HRP
The hapten BFNH was covalently attached to HRP using the mixed-anhydride method mentioned
above.
Preparation of standard solutions
Standard solutions were prepared on glass tubes instead of the plastic tubes, since it was found that
-3
5
the plastic tubes adsorbed carbofuran. Standards from 2 ×10 to 2 ×10 ng/mL were prepared by 1/10
dilution in PBST (8.03 mmol Na HPO , 1.97 mmol KH PO , 137 mmol NaCl, 2.68 mmol KCl,
containing 0.05% Tween-20, pH 7.4). The final carbofuran concentrations in the assay ranged from 1
2
4
2
4
-3
5
×
10 to 1 × 10 ng/mL.
The development of ELISAs for carbofuran
Flat-bottom polystyrene ELISA plates were coated overnight with conjugate solution of 50 mmol/L
carbonate buffer (pH 9.6). A volume of 50 μL per well was used throughout all assay steps and all
incubations were carried out at 37°C.
Conjugate-coated indirect competitive ELISA (Format I). Plates were coated with BFNH-OVA
conjugates of 0.5 ng/mL previously determined to be the optimal by checkerboard titration.
Carbofuran standards (50 μL) were added into the well and 50 μL antibody (0.057 ng/mL) was added
and then incubated for 2 h. Finally, the plates were incubated for 1 h with peroxidase-labeled goat anti-
mouse immunoglobulins diluted in PBST solution. Peroxidase activity bound to the wells was

Molecules 2008, 3
879
determined by adding the substrate solution (2 mg/mL o-phenylendiamine and 0.012% H O in 25
2
2
mmol citrate, 62 mmol sodium phosphate, pH 5.4). After 15 min, the reaction was stopped with 2.5
mol/L sulfuric acid. The absorbance was recorded at 490 nm. Competitive curves were obtained by
plotting the inhibition rate against the logarithm of carbofuran concentration. Sigmoidal curves were
fitted to four-parameter logistic equation, using the Originpro 7.5 software package.
A490 max − A490
Inhibition rate (%) =
× 100
(4)
A490 max
Conjugate-coated direct competitive ELISA (Format II). In this format, plates were coated with
BFNH-OVA (0.25 ng/mL) conjugates. The competitive ELISA was established for 0.5 h between
carbofuran standards and the selected concentration (1:80) of antibody-HRP. Peroxidase activity was
measured using the method mentioned above.
First antibody-coated direct competitive ELISA (Format III). Plates were coated with antibody (0.35
ng/mL) for carbofuran. The competitive ELISA was established for 2 h between carbofuran standards
and the selected concentration (1:800) of BFNH-HRP. Peroxidase activity was measured using the
method mentioned above.
Second antibody-coated direct competitive ELISA (Format IV). Plates were coated with rabbit anti-
mouse immunoglobulins (1 μg/mL). The selected concentration (1.42 ng/mL) of antibody for
carbofuran was added and then incubated for 0.5 h. The competitive ELISA was established for 1 h
between carbofuran standards and the selected concentration (1:400) of BFNH-HRP. Peroxidase
activity was measured using the method mentioned above.
Preparation of samples
Lettuce and cabbage samples were obtained from the market of the South China Agriculture
University (SCAU) before use. The lettuce and cabbage juice samples were prepared with a fruit
pulping machine and diluted l:5 in PBS and then spiked with carbofuran at 0.1, 1, 10 and 50 ng/mL.
The water samples from pond and soil samples from vegetable plot were obtained from the campus of
SCAU. The soil samples (50 g) were diluted with PBS (50 mL) and stirred for 12 h. The clear
supernatant was collected by centrifugation at 4,000 rpm for 10 min. The soil supernatant and water
samples were spiked with carbofuran at 0.1, 1, 10 and 50 ng/mL.
Acknowledgements
th
This work was supported by the National S&T 10 Five-Year Plan of China, Grant No.
2
002BA518A06-04, Guangdong Province Key Project for Natural Science, Grant No. 036842,
Guangzhou Key Project for Science and Technology, Grant Nos. 2004Z2-E0031 and 2004Z1-E0061,
Natural Science Foundation of Guangdong Province, Grant No. 06300421, the National High-Tech

Molecules 2008, 3
880
Research, Development Program of China (863 Program), Grant No. 2006AA10Z447, and the
Knowledge Innovation Program of the Chinese Academy of Sciences, Grant No. KSCX2-YW-N-036.
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1
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Sample Availability: Available from the authors.
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