Immunoassay development for the class-specific assay for types I and II pyrethroid insecticides in water samples

Article abstract of DOI:10.3390/molecules15010164

Five generic haptens of pyrethoid insecticides, which were classified as three types, were designed and synthesized: the first (hapten 1) is for type I pyrethroids without a cyano group, the second (hapten 2 and XQ) for type II pyrethroids with a cyano group, and the third (hapten 4 and 5) for both types of pyrethroids with loss of the ester group. The hapten structures were confirmed by MS and ¹H-NMR. Hapten 1 and 2 were conjugated with BSA respectively and haptens 1-5 were conjugated with OVA. Four polyclonal antisera were raised against BSA conjugates including a mixture conjugate, and twenty antibody/coating conjugate combinations were selected for studies of assay sensitivity and specificity for pyrethroids. The study revealed the best combination, which showed equal high sensitivities (I₅₀ is around 0.02 μg mL^-1) to both types of pyrethroids. The immunity results suggest that, with a mixture conjugates, a polyclonal antibody against a group of insecticides can be prepared for multi-residue assays.

Full text of DOI:10.3390/molecules15010164

Molecules 2010, 15, 164-177; doi:10.3390/molecules15010164
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Immunoassay Development for the Class-Specific Assay for
Types I and II Pyrethroid Insecticides in Water Samples
Qi Zhang, Wen Zhang, Xiuping Wang and Peiwu Li *
Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China;
E-Mails: zhangqi521x@yahoo.cn (Q.Z.); zhangwen@oilcrops.cn (W.Z.);
xiupinwang@sina.com (X.P.W.)
* Author to whom correspondence should be addressed: E-Mail: peiwuli@oilcrops.cn.
Received: 28 October 2009; in revised form: 10 December 2009 / Accepted: 29 December 2009 /
Published: 4 January 2010
Abstract: Five generic haptens of pyrethoid insecticides, which were classified as three
types, were designed and synthesized: the first (hapten 1) is for type I pyrethroids without
a cyano group, the second (hapten 2 and XQ) for type II pyrethroids with a cyano group,
and the third (hapten 4 and 5) for both types of pyrethroids with loss of the ester group.
1
The hapten structures were confirmed by MS and H-NMR. Hapten 1 and 2 were
conjugated with BSA respectively and haptens 1-5 were conjugated with OVA. Four
polyclonal antisera were raised against BSA conjugates including a mixture conjugate, and
twenty antibody/coating conjugate combinations were selected for studies of assay
sensitivity and specificity for pyrethroids. The study revealed the best combination, which
showed equal high sensitivities (I50 is around 0.02 µg mL-1) to both types of pyrethroids.
The immunity results suggest that, with a mixture conjugates, a polyclonal antibody
against a group of insecticides can be prepared for multi-residue assays.
Keywords: hapten; class-specific antibody; sensitivity; immunoassay; pyrethoid
insecticides
Abbreviations: THF, tetrahydrofuran; DMF, dimethyl formamide; NHS, N-
hydroxysuccinimide; DCC, N,N-dicyclohexylcarbodiimide; BSA, bovine serum albumin;
OVA, ovalbumin; CR, cross-reactivity; ELISA, enzyme-linked immunosorbent assay;
TMB, Tetramethylbenzidine; GAM-HRP, peroxidase-labeled goat anti-mouse
immunoglobulins; I50, concentration giving 50% inhibition of maximum response; LOD,

Molecules 2010, 15
165
limit of detection; PBS, 150 mM phosphate buffer, pH 7.4; PBST, PBS containing 0.05%
(v/v) Tween 20
Introduction
Pyrethroid insecticides have been being used widely from agricultural uses [1-4] to home pest
control [5,6] and are effective against a broad range of pests. The synthetic pyrethroids and natural
pyrethrins can be divided into two groups of compounds on the basis of their chemical structure and
mechanism of action at insect target sites (Figure 1): The type I compounds are simple cyclic alcohol
esters of 2,2-dimethyl-3-(2-methyl-1-propenyl)-cyclopropanecarboxylic acid, and the type II
compounds are esters of an aryl cyanohydrin [7]. Synthetic pyrethroid residues have been seen in
agricultural products [8,9] and food [10]. Lower and lower and lower amounts are being allowed
worldwide by regulatory agencies [11]. Many pyrethroids act as a neurotoxin, they are also highly
toxic to aquatic life, particularly fish [12,13], so it is very important to develop a rapid, sensitive,
specific methods to monitor pyrethroid residues in food products.
Figure 1. Structures of some synthetic pyrethroids.
Type I pyrethoids
Type II pyrethoids
Br
Br
O
O
O
O
O
CN
O
deltamethrin
phenomethrin
Cl
Cl
Cl
Cl
O
O
O
O
O
CN
O
cypermethrin
permethrin
F3C
Cl
O
O
O
CN
cyhalothrin
Current analytical methods for pyrethroid insecticides involve multistep sample cleanup procedures
followed by gas chromatography on instruments equipped with an electron capture detector (GC-EC)
[14,15], gas chromatography-mass spectrometry (GC-MS) [16,17], liquid chromatography combined
with postcolumn fluorimetry derivatization and fluorescence detection (HPLC-FD) [18,19], or high
performance liquid chromatography-mass spectrometry (HPLC-MS) [20]. These methods meet the
requirements for sensitivity and accuracy of pyrethroids measurements. On the other hand, they are

Molecules 2010, 15
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relatively intensive, time-consuming, expensive, and not particularly suitable for large numbers of
samples.
Immunoassays have been considered as a valuable supplement to existing, and rapidly developing,
chromatographic techniques, because they have attractive features including high sensitivity and
selectivity, rapid detection, and the possibility of analysis of difficult matrices without extensive pre-
treatment [21].
When an immunoassay for a single analyte was developed in the past years, people all expected
CRs as lower as possible [22–27]. With the increase in requests for detection of chemical residues,
there has been a gradual focus on total and multi-residues immunoassays. So, with higher CRs, some
immunoassays for a type of compounds are also expected and some class specific antibodies against
organophosphorus [28–30], type I or type II pyrethoid insecticides have been reported [7,31,32]. Here
we aimed to develop a combination of coating conjugates and antibodies with equal high sensitivity to
both type I and type II pyrethoids.
Scheme 1. Synthetic schemes for the preparation of generic haptens 1, 2, 4, and 5 and the
structure of hapten XQ.
O
O
O
OH
O
COOH
O
O
dichloromethane
NaCN / HCl
O
O
hapten 1
OH
CHO
O
CN
O
O
O
O
COOH
O
CN
O
dichloromethane
hapten 2
Br(CH2)2COOC2H5
O
OH
DMF
O
COOC2H5
O
NaOH
O
O
COOH
O
hapten 4
O
O
O
COOH
O
N
DCC / NHS
DMF
O
O
O
COOH
NH2CH2CH2COOH
N
H
hapten 5
O
HN
COOH
Br
Br
O
O
O
CN
hapten XQ

Molecules 2010, 15
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Results and Discussion
Hapten design and conjugate verification
Obtaining haptens with proper chemical structures is the key to development of novel antibodies
and immunoassays. According to the characteristics of the two types of synthetic pyrethroid pesticides
[32], here we designed three types of haptens (Scheme 1): the first (hapten 1) is for type I pyrethroids
without a cyano group, the second (hapten 2 and XQ) for type II pyrethroids with a cyano group, and
the third (hapten 4 and 5) for both types of pyrethroids with loss of the ester group. Most of these
haptens were synthesized simply with just one or two steps and without any rigorous condition.
Through scanning the UV-Vis spectrum, hapten/protein ratios were calculated by measuring the
absorbance of the hapten, the protein, and the hapten-protein conjugate at the same wavelength, and
the ratios of BSA-hapten 1, BSA-hapten 2, OVA-hapten 1, 2, 4, 5, XQ were 14, 13, 9, 6, 8 and 4.
Here, hapten 1 and hapten 2 were used as immunizing haptens. To attempt to produce specific
antibodies against both types of pyrethroid insecticides, a mixture of conjugates of hapten 1 and
hapten 2 was also regarded as an immunogen. All of the above OVA conjugates, (OVA-hapten 1
(CAg-1), OVA-hapten 2 (CAg-2), OVA-hapten 4 (CAg-4), OVA-hapten 5 (CAg-5), OVA-hapten XQ
(CAg-XQ)) were used as coating antigen. According to references, some generic immunizing haptens
of each type of pyrethroids have been described [7,31–33]. Comparing with the reported, both hapten 1
and hapten 2 retain the ester group but lose a cyclopropane group.
Effect of the haptens on affinity of the antisera for the coating antigens
The most important precondition is that a suffcient titer value for combination of antibody and
antigen exist [34]. To investigate homo- and heterologous affinity, twenty antibody/coating conjugate
combinations were tested by a noncompetitive ELISA protocol. Some titer values, which were found
to be difference among the combinations are shown in Table 1.
Table 1. The titer values of antisera (absorbance at 450 nm). a
Coating antigen (1 μg mL-1)
Antibody
dilution
Immunogen
Antiserum
cAg-1
1.65 1
0.573
1.344
1.302
cAg-2
0.398
1.484
0.925
1.064
cAg-4
0.629
0.384
0.754
0.400
cAg-5
0.412
0.198
0.458
0.352
cAg-XQ
0.257
0.626
0.174
0.174
BSA-hapten 1
BSA-hapten 2
BSA-hapten 1
pAb-1 b
pAb-2 b
1: 8000
1: 8000
1: 3200
1: 3200
pAb-m1 c
pAb-m2 c
+ BSA-hapten 2
a
Four antibodies, named pAb-1, pAb-2, pAb-m1, and pAb-m2 respectively, were determined.
Absorbances were measured by a checkerboard pattern with several coating conjugate
concentrations and several antibody dilutions, and measured after a 12-min incubation with TMB at
37 °C. For convenience, only data from a coating antigen concentration of 0.1 μg per well and an
antibody dilution of 1:8,000 or 1:3,200 are shown. Titer values are the means of three replicates.
The results indicated that the antibodies induced by type I hapten (hapten 1) had cross-reactivity to
type II hapten (hapten 2) and the reverse was also true. Considering the distguishing titer difference
between coating haptens with and without a cyano group, we thought that the cyano group had a

Molecules 2010, 15
168
strong effect on such a antibody/hapten reaction system. We also discovered significantly different
data for the combination of pAb-2/cAg-1 (0.573) and of pAb-2/cAg-4 (0.384), which indicated that the
ester group had also little effects on the reaction system.
The results of those antibodies against the mixture immunogen (BSA-hapten 1 and BSA-hapten 2)
showed that the combination of pAb-m1/cAg-4 had a high titer value (0.745) in addition to “semi-
homologous combinations”, such as pAb-m1/cAg-1, pAb-m1/cAg-2 and so on. Those combinations of
antibody/coating antigen, with enough affinity and possibility to develop an ELISA for both types of
synthetic pyrethroid insecticides, were selected for further studies.
Effect of the haptens on ELISA sensitivity and specificity
To investigate homo- and heterologous sensitivity, five antibody/coating conjugate combinations
were tested by a noncompetitive ELISA protocol. To screen the broadest specific combination, five
synthetic pyrethroid insecticides were select for cross-reactivity analysis. The sensitivity and
specificity data are shown in Table 2.
In the first combination of pAb-1/cAg-2, the ELISA was only sensitive to type I pyrethroids
(phenomethrin and permethrin). Nevertheless the ELISA showed more sensitivity to type II
(datamethrin, cypermethrin, and cyhalothrin) than to type I in the second combination of pAb-2/cAg-1.
Considering that pAb-1 was prepared with type I immunogen and that pAb-2 was prepared with type
II, we could obviously see the cyano group on the type II immunogen had an important effect during
the immunization process. The result suggested that, with only one type pyrethroid immunogen,
whether type I or type II, it is difficult (even impossible) to obtain antibodies with equal sensitivity to
both types of pyrethroids.
Therefore, to obtain the antibodies with equal sensitivity to both types of pyrethroids, a mixed
immunogen with type I and type II pyrethroid conjugates was used for antibody preparation. In Table
2, the last three combinations were all against such mixed immunogen. The ELISA of pAb-m1/cAg-1
combination showed an I50 range of 0.1–0.3 µg mL-1, pAb-m1/cAg-2 combination 0.2–0.6 µg mL-1,
and pAb-m2/cAg-4 combination 0.016–0.023 µg mL-1. According to these results we found the last
combination of pAb-m2/cAg-4, with a heterologous ELISA system, had the most equal sensitivity (all
I50s were around 0.02 µg mL-1) to both types of pyrethroids, and the sensitivity was close to the
references (0.02–0.03 µg mL-1 for type I, 0.0015–0.013 µg mL-1 for type II) [7,31,32]. So the
combination of pAb-m2/cAg-4 was select for further research.
Table 2. Specificity of antibody/coating antigen combinations.
Antibody/coating
antigen
I50
Cross-reactivity
%
100
Pesticide
b
µg mL-1
0.29
phenomethrin
permethrin
0.42
69.0
<0.1
<0.1
<0.1
100
62.8
392.3
416.3
351.7
c
pAb-1/cAg-2
pAb-2/cAg-1
datamethrin
cypermethrin
cyhalothrin
phenomethrin
permethrin
datamethrin
cypermethrin
cyhalothrin
-
-
-
c
c
0.204
0.325
0.052
0.049
0.058

Molecules 2010, 15
169
Table 2. Cont.
phenomethrin
permethrin
datamethrin
cypermethrin
cyhalothrin
phenomethrin
permethrin
datamethrin
cypermethrin
cyhalothrin
phenomethrin
permethrin
(Trans/Cis)
permethrin
(Trans)
0.278
100
0.333
0.136
0.098
0.165
0.247
0.324
0.495
0.631
0.386
0.017
83.5
204.4
283.7
168.5
100
76.2
49.9
39.1
64.0
100
pAb-m1/cAg-1
pAb-m1/cAg-2
0.022
0.048
77.3
35.4
datamethrin
cypermethrin
cyhalothrin
esfenverate (1S,2S)
fenverate (1R/S,2R/S)
Tetramethrin
Bifenthrin
0.023
0.019
0.016
0.186
0.125
>10
73.9
89.5
106.2
9.1
13.6
<0.1
<0.1
<1
pAb-m2/cAg-4
>10
>1
3-phenoxybenzoic acid
a
b
The coefficient of variation(CV) was below 12%; The cross-reactivity of phenomethrin in all combinations was
regarded as 100%, and those of other pyrethroids were calculated as follows: cross-reactivity (%) = [I50 (phenomethrin)
/I50 (other pyrethoid)] × 100; c The data was not calculated because no significant inhibition was observed.
Average competitive curve and spiked sample analysis
In the above selected ELISA system, the coating antigen cAg-4 and the polyclonal antibody pAb-
m1 were used. A series of concentrations of each standard pyrethroid: phenomethrin, permethrin,
datamethrin, cypermethrin, and cyhalothrin was tested, whose results contributed to the average
standard curve (Figure 2). Among the results, the average coefficient of variation (CV) was 16%, I50
was 0.02 µg mL-1, and the dynamic range (I20-I80) was 0.002–0.084 µg mL-1.
With the average standard curve, a spiking experiment was carried out for an elementary accuracy
evaluation. The results showed the recoveries obtained by standard pyrethroids added to tap water
samples were from 57% to 73% (Table 3), which indicated that the combination of pAb-m2/cAg-4 will
be a useful screening test system for both type I and type II pyrethroids.
Theoretically, we also paid attention here to whether the total concentration of several different
compounds with a same type could be calculated as that of just one analyte in a class specific ELISA
curve. However, the results could not give clearly an answer to this, so we need to do more related
work to answer this question.

Molecules 2010, 15
Figure 2. Average standard inhibition curve of five pyrethroids. In an ELISA system, the
170
coating antigen cAg-4 was used with a concentration of 2 µg mL-1, and the polyclonal
antibody pAb-m1 was used with a dilution of 1:4,000. Then a series concentrations of each
standard pyrethroid phenomethrin, permethrin, datamethrin, cypermethrin, and cyhalothrin
was tested, whose results contributed to the average standard curve (the average maximum
absorbance was 0.952, and the slope value is -1.266). And the average coefficient of
variation (CV) was 16%, I50 was 0.02 µg mL-1, and the dynamic range (I20-I80) was
0.002–0.084 µg mL-1.
100
80
60
40
20
0
0.0001
0.001
0.01
0.1
1
10
Concentration of pyrethroids (µg mL-1)
Table 3. Recovery test of synthetic pyrethroids in drinking water.
pyrethroid
insecticide
spiked
(µg mL-1)
0
theoretical
(µg mL-1)
0
found
(µg mL-1)
< 0.002
average recovery
± SD (%)
-
permethrin
datamethin
permethrin
datamethrin
cyhalothrin
permethrin
datamethin
cyhalothrin
phenomethrin
permethrin
datamethin
cyhalothrin
phenomethrin
cypermethrin
0.01
0.02
0.01
0.02
0.02
0.02
0.04
0.04
0.10
0.02
0.04
0.04
0.10
0.20
73.3 ± 14.2
0.006
0.004
56.9 ± 10.6
72.9 ± 6.9
0.010
0.006
0.040
0.080
0.029
0.053
66.3 ± 13.2

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Experimental
Reagents and materials
Chemical reagents for hapten synthesis and pesticide standards used for cross-reactivity studies
were supplied by China Redsun Group Nanjing No.1 Pesticide Co., Ltd. (Nanjing, China) and Jiangsu
Pesticide Research Institute (Nanjing, China). Analytical-grade solvents were from Sinopharm Group
Chemical Reagent Co., Ltd. (Shanghai, China). Tween 20, N-hydroxysuccinimide (NHS), N,N-
dicyclohexylcarbodiimide (DCC), tetramethylbenzidine (TMB), peroxidase-labeled goat anti-rabbit
immunoglobulins (GAM-HRP), bovine serum albumin (BSA), ovalbumin (OVA), and complete and
incomplete Freund’s adjuvants were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other
reagents were analytical grade. Glass sheets precoated 0.3-mm silica gel 60 F254 for thin-layer
chromatography (TLC) and silica gel (60–230 mesh) for column chromatographic purifications were
purchased from Qingdao Haiyang Chemical Co., Ltd (Qingdao, China).
Instruments
1
Mass spectra were obtained on a HPLCMS-LTQ XL spectrometer (Thermo-Fisher, USA). H-
nuclear magnetic resonance (NMR) spectra were obtained with an INOVA-600 MHz spectrometer
(Varian, Palo Alto, CA, USA). Chemical-shift values were given in parts per million (ppm) downfield
from the internal standard deuterium chloroform. Coupling constants are expressed in Hz and the
abbreviations s, d, t, m, and Ar represent singlet, doublet, triplet, multiplet, and aromatic, respectively.
UV-Vis spectra were recorded on a Beckman 640 spectrophotometer. Polystyrene 96-well microtiter
plates were from Costar (Corning, MA, USA). A Wellwash 4MK-2 microplate strip washer (Thermo
Electron Corporation.) was used to wash ELISA plates. Absorbance (A) was measured using a
microplate reader (Wallac 1420 Victor 3, Perkin Elmer Inc.).
Hapten synthesis and verification
Five haptens were mentioned in this paper. The synthetic routes for haptens 1, 2, 4, and 5 are
illustrated in Scheme 1. The structure of our previously prepared hapten XQ [35] is also shown in
Scheme 1.
4-Oxo-4-(3-phenoxybenzyloxy)butanoic acid (hapten 1): Succinic anhydride (0.7 g, 6 mmol) was
added to a solution of (3-phenoxyphenyl)methanol (1.2 g, 6 mmol) in dichloromethane (100 mL).
After stirring at room temperature overnight, the solution was washed with water and then dried over
anhydrous sodium sulfate. Finally, the solution was concentrated and gave hapten 1 (1.6 g, 94%) as a
1
white solid. H-NMR (CD3Cl) δ: 2.66-2.68 (t, 2H, CH2), 2.69-2.70 (t, 2H, CH2), 6.94-7.36 (m, 9H,
Ar); MS (ESI) m/z (%): 285 (M-H+, 89).
4-(Cyano(3-phenoxyphenyl)methoxy)-4-oxobutanoic acid (hapten 2): The aldehyde 3-
phenoxybenzaldehyde (1.2 g, 6 mmol), in THF (9 mL) and water (1 mL), was cooled in ice. Powdered
potassuim cyanide (0.2 g, 6 mmol) was added into the solution of the aldehyde. With stirring, 24 N
HCl (0.4 mL) was dropped slowly into the mixture. The reaction went on for 40 min, and then the

Molecules 2010, 15
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mixture was acidified with 3 N HCl and extracted with ether, and the organic phase was washed with
water, dried over anhydrous magnesium sulfate, and evaporated to give the cyanohydrin, 2-hydroxy-2-
(3-phenoxyphenyl)acetonitrile, as a brown oil. Subsequently the brown oil was used to synthesize
hapten 2 by the same method as used for hapten 1. Finally, 1.6 g of hapten 2 was obtained as a white
solid, giving a yield of 87%. 1H-NMR (CDCl3) δ: 2.68-2.70 (t, 2H, CH2), 2.72-2.75 (t, 2H, CH2), 5.30
(s, 1H, Ar-CH), 7.03-7.41 (m, 9H, Ar); MS (ESI) m/z (%): 310 (M-H+, 62).
3-(3-Phenoxybenzyloxy)propanoic acid (hapten 4): To a solution of (3-phenoxyphenyl)methanol
(1.0 g, 5 mmol) in dry acetone (30 mL) were added anhydrous potassium carbonate (0.6 g, 5 mmol)
and ethyl bromoacetate (0.8 g, 5 mmol). After refluxing for 15 h, the mixture was filtered and the
solvent was evaporated under reduced pressure. The residue was dissolved in ethyl acetate, washed
with cold water, 1 M NaOH and 4 M NaCl, and dried over anhydrous sodium sulfate. Removal of the
solvent gave ethyl 3-(3-phenoxybenzyloxy)propanoate as a yellow oil. The ester was dissolved in THF
(2 mL) and 1 M NaOH (12 mL) was added. After refluxing for 2 h, the mixture was extracted with
CH2Cl2. The aqueous layer was acidified to pH 3-4 by careful addition of concentrated HCl and then
extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and
concentrated to give hapten 4 (0.7 g, 52%) as a white solid. 1H-NMR (acetone-d6) δ: 2.01-2.02 (t, 2H,
CH2-O), 2.16-2.17 (t, 2H, CH2-C), 3.38 (s, 2H, Ar-CH2), 6.99-7.39 (m, 9H, Ar); MS (ESI) m/z (%):
285 (M-H+, 66).
3-(3-Phenoxybenzamido)propanoic acid (hapten 5): In a solution of anhydrous DMF (3 mL),
3-phenoxybenzoic acid (1.1 g, 5 mmol), NHS (0.6 g, 5 mmol) and DCC (1.0 g, 5 mmol) were added.
With stirring, the reaction went on for 3 h at room temperature. The mixture was stored at 4 °C
overnight, then the supernatant containing the active ester, 2,5-dioxopyrrolidin-1-yl
3-phenoxybenzoate, was separated and slowly added dropwise to a solution of 3-aminopropanoic acid
(0.5 g, 5 mmol) in PBS (12 mL). The reaction was carried on for 1 h at room temperature and then
overnight at 4 °C. After removal of the solvent, the residue was dissolved in ethyl acetate, washed with
cold water, 1 M NaOH and 4 M NaCl, and dried over anhydrous sodium sulfate. Final removal of the
1
solvent gave hapten 5 (0.8 g, 57%) as a white solid. H-NMR (acetone-d6)δ: 2.24-2.27 (m, 1H, CH2),
2.02-2.06 (t, 2H, CH2-C), 7.05-7.79 (m, 9H, Ar), 10.83 (s, 1H, NH); MS (ESI) m/z (%): 284
(M-H+, 32).
Preparation of immunogens and coating antigens
The conjugations of the five haptens above and the estimations of hapten densities were carried out
according to Zhang [36]. To generate immunogens, haptens 1 and B were covalently attached through
their carboxylic acid moieties to the lysine groups of BSA using the active ester method. Using the
same method, haptens 1, 2, 4, 5, and XQ were coupled to OVA to obtain coating antigens. The
immunogens and coating antigens were purified by dialysis in phosphate buffer (PB: 0.02 mol L-1,
pH 6.8). The conjugates were stored at -20 °C until use. UV-Vis spectral data were used to confirm the
structures of the final conjugates. Assuming that the molar absorptivity of haptens was the same for the
free and conjugated forms, the hapten densities (the number of hapten molecules per molecule of
protein) of the conjugates were estimated directly by the mole absorbance ε:

Molecules 2010, 15
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Hapten density = (ε conjugation -ε protein )/ε hapten
Immunization
Four female New Zealand white rabbits of about three months of age were immunized with the
conjugates of BSA-hapten 1, BSA-hapten 2, or the mixture of equal amount of BSA-hapten 1 and
BSA-hapten 2. The first dose consisted of 800 μg of conjugate injected as an emulsion of PBS and
Freund’s complete adjuvant. Three subsequent injections emulsified in Freund’s incomplete adjuvant
were given at three-week intervals. One week after the last injection, the rabbits were bled, and the
production of Ab was made following the protocol reported by Shan [23]. The anti-hapten antibody
titers of the sera were tested by indirect ELISA, and the analyte recognition properties were examined
by competitive indirect ELISA. Four sera were obtained and tested: pAb-1 against BSA-hapten 1,
pAb-2 against BSA-hapten 2, and pAb-m1 and pAb-m2 against the mixture immunogen.
Titration of antisera
The titers of antisera were determined by measuring the binding of serial dilutions of the antisera to
the corresponding coating antigen (hapten-OVA) using noncompetitive ELISA protocol. Polystyrene
microtiter plates were coated with the coating antigen (1 μg mL-1, 50 μL per well) in 50 mmol L-1
carbonate-bicarbonate buffer (pH 9.6) by 2 h incubation at 37 °C. The following steps were the same
as the description of Zhang [33]. The coated plates were washed five times with PBST (PBS
containing 0.05% Tween 20, pH 7.4) and blocked by incubation with 1% gelatin in PBS (100 μL per
well) for 1.5 h at 37 °C. After another washing step, 50 μL per well of antiserum diluted with PBS
(1:1,600–1:512,000) were added to the plate, and the plates were incubated for 1 h at 37 °C. After
another washing step, 50 μL of a GAM-HRP conjugate diluted 1:10,000 with PBST were added to
each well and incubated for 1 h at 37 °C. Next, the plates were washed again, and 50 μL of substrate
solution (3.3 μL of 30% H2O2 and 400 μL of 0.6% TMB in DMSO per 25 mL of acetate buffer,
pH 5.5) were added to each well. Color development was stopped after 15-30 min with 25 μL per well
of 2 mol L-1 H2SO4. The absorbance was measured using the single-wavelength mode at 450 nm.
Determination of the effect of the haptens on the affinity of antisera for coating antigens
The affinity of each of the six antisera for each of the five coating antigens (hapten-OVA
conjugates) was determined by noncompetitive indirect ELISA as follows: all incubations were
performed at 37 °C, including the incubation of 2 h with the coating antigens. Microtiter plates were
coated with the hapten-OVA conjugates (25, 50, 100, or 200 ng per well), and 50 μL of antiserum was
diluted with PBST (1:1,600, 1:3,200, 1: 6,400, 1:8,000, 1:12,800, or 1:16,000). The other steps were
described as the above.
Determination of the effect of the haptens on the ELISA sensitivity and specificity
The effect of hapten heterology between the immunogen and the coating antigen on the ELISA
sensitivity and specificity was investigated by competitive indirect ELISA for all possible
combinations of antiserum and coating antigen. The assay procedure was presented previously [34].

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All incubations were performed at 37 °C except for the incubation with the coating antigens.
Microtiter plates were coated with hapten-OVA conjugate (1 μg mL-1, 50 μL per well) in 50 mM
carbonate-bicarbonate buffer (pH 9.6) by overnight incubation at 4 °C. The plates were washed five
times with PBST and blocked by incubation with 100 μL per well of 1% gelatin in PBS for 1.5 h. After
another washing step, 25 μL per well of serial dilutions of the analyte in 40% methanol-PBS were
added, followed by 25 μL per well of antiserum diluted in PBST. After incubation for 1 h, the antibody
binding was assessed as described above using HRP-conjugated goat anti-mouse IgG diluted in PBST.
I50 values (the concentration at which the binding of the antibody to the coating antigen is inhibited by
50%) were determined using logistic equations. The specificities of the ELISAs against several
pyrethroid pesticides were determined and calculated as follows: CR (%) = [I50 (fenthion)/I50 (test
compound)] × 100.
Average competitive curve and water sample analysis
In an ELISA system, the coating antigen cAg-4 was used with a concentration of 2 µg mL-1, and the
polyclonal antibody pAb-m1 was used with a dilution of 1:4000. Then a series concentrations (0.0001,
0.001, 0.01, 0.1, 1, and 10 µg mL-1) of each standard pyrethroid phenomethrin, permethrin,
datamethrin, cypermethrin, and cyhalothrin was tested, whose results contributed to the average
standard curve.
To simply evaluate the above ELISA, several water samples spiked with different pyrethoid
insecticides were prepared. The tap water was collected from local families in Wuhan, Hubei Province.
For the spike-and-recovery test, five final concentrations (0, 0.03, 0.05, 0.2, 0.4 µg mL-1) of
pyrethroids of the above samples were prepared. These Water samples were detected directly by the
developed ELISA (the samples were diluted five times with PBS-methanol buffer).
Conclusions
Usually, a generic hapten was used for preparation of a class specific antibody against a type of
anlytes [37]. In this paper, with a mixture of conjugates of type I and type II pyrethroids, we prepared a
polyclonal antibody against both types of pyrethroid insecticides. Therefore, use of a mixture
immunogen, seems to provide a new behavior for preparation of class specific antibodies against a
group of small molecular analytes. In addition, we found the best combination of pAb-m2/cAg-4 with
equal high sensitivities (about 0.02 µg mL-1) to both types of pyrethroid insecticides tested here, such
as phenomethrin, permethrin, datamethrin, cypermethrin, and cyhalothrin. With the developed
heterologous ELISA system, the recovery tests show that this assay can be used for screening water
samples for pyrethroid multi-residues.
Acknowledgements
This work was supported by the Chinese National ‘863’ High-Tech. Research Program
(2006AA10Z448), the Program of National Science Foundation of China (30800771), and by projects
of Science and Technology of Wuhan (200820337086, 200950431224) and Special Foundation of
President of the Chinese Agricultural Academy of Sciences.

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