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General immunoassay for pyrethroidsbased on a monoclonal antibodyXiujin Chenab, Liguang Xua, Wei Maa, Liqiang Liua, Hua Kuanga,Libing Wangc & Chuanlai Xua

a School of Food Science & Technology, State Key Laboratory ofFood Science & Technology, Jiangnan University, Wuxi, Chinab Food and Biological Engineering Institute, Henan University ofScience and Technology, Luoyang, Chinac Research Centre of Hunan Entry-Exit Inspection and QuarantineBureau, Changsha, ChinaPublished online: 02 May 2013.

To cite this article: Xiujin Chen, Liguang Xu, Wei Ma, Liqiang Liu, Hua Kuang, Libing Wang &Chuanlai Xu (2014) General immunoassay for pyrethroids based on a monoclonal antibody, Food andAgricultural Immunology, 25:3, 341-349, DOI: 10.1080/09540105.2013.794328

To link to this article: http://dx.doi.org/10.1080/09540105.2013.794328

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General immunoassay for pyrethroids based on a monoclonal antibody

Xiujin Chena,b, Liguang Xua, Wei Maa, Liqiang Liua, Hua Kuanga*, Libing Wangc

and Chuanlai Xua

aSchool of Food Science & Technology, State Key Laboratory of Food Science & Technology,Jiangnan University, Wuxi, China; bFood and Biological Engineering Institute, Henan Universityof Science and Technology, Luoyang, China; cResearch Centre of Hunan Entry-Exit Inspectionand Quarantine Bureau, Changsha, China

For the development of class-specific antibody against pyrethroids, four haptenswere designed and synthesised. A hybridoma 3E9 was successfully selected forproduction of antibody, which originated from mouse immunised with conjugatesof hapten 1 [(RS)-cyano-3-phenoxybenzyl-2, 2, 3, 3-tetramethylcyclopropane-carboxylic acid] and keyhole limpet hemocyanin. A sensitive general immunoas-say was developed with a monoclonal antibody from 3E9 cell line and coatingantigen from conjugates of hapten 4 [N-(3-phenoxybenzoyl)-4-amino-L-pheny-lalanine] and bovine serum albumin. Under the optimised conditions, theantibody has good recognition to cypermethrin with 50% inhibition concentra-tion (IC50) value of 1.790.76 ng mL�1. The cross-reaction to analogues wascalculated as 12% for fenpropathrin, 4% for esfenvalerate. Besides, the antibodyshowed affinity to bifenthrin, deltamethrin and fenvalerate in different degreeswith IC50 value ranging from 191.8 to 298.5 ng mL�1. The performance of thisimmunoassay was evaluated by fortified real water samples with three represen-tative compounds. Recoveries were 76�118%, and the results showed that thisimmunoassay could be applied to monitor pyrethroid residues.

Keywords: pyrethroid; immunoassay; monoclonal antibody; water

Introduction

The synthetic pyrethroids are used widely in agricultural production for their

remarkably high insecticidal activity and low mammalian toxicity compared with

organochlorinated and organophosphate compounds (Class & Kintrup, 1991).

However, due to the extensive human use and release of synthetic pyrethroids,

more and more potential toxic effects from them on ecological environment and

human health have been raised (Kuivila et al., 2012; Palmquist, Fairbrother, Salatas,

& Guiney, 2011). Long-time exposure to synthetic pyrethroids leads to endocrine

disruption (El-Magd, Sabik, & Shoukry, 2011), reproductive toxicity (Wang, Chen,

Zhang, & Fang, 2009) and impairment of organs.Current analysis of synthetic pyrethroids is to employ chromatography techni-

ques such as gas chromatography (GC) with an electron capture detector (ECD)

(Regueiro, Llompart, Garcia-Jares, & Cela, 2007), GC with flame ionisation detector

(FID) (Pinheiro & de Andrade, 2009), liquid chromatography�mass spectrometry

(LC-MS) (Dulaurent, Moesch, Marquet, Gaulier, & Lachatre, 2010; Galera, Garcia,

*Corresponding author. Email: khecho@163.com

Food and Agricultural Immunology, 2014

Vol. 25, No. 3, 341�349, http://dx.doi.org/10.1080/09540105.2013.794328

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& Valverde, 2006) and LC combined with fluorescence detector (Bagheri, Ghanbar-

nejad, & Khalilian, 2009). A longstanding shortcoming of the above measure lies in

its complexity in sample preparation and time-consuming process. An increasing

number of detection methods developed in recent years have been used for pyrethroidresidue determinations including immunoassay and biosensors (Ahn et al., 2007).

Immunoassay, especially enzyme-linked immunosorbent assay (ELISA), has been

reported (Hao et al., 2009). Most of these immunoassays were based on polyclonal

antibodies and seldom reports are found to develop a broad specific detection of

pyrethroids using monoclonal antibodies (mAbs).

In this study, we obtained a general monoclonal antibody that shows affinity to

multi-pyrethroids with satisfactory IC50 values. The proposed ELISA was success-

fully used to monitor pyrethroids in local lake water.

Materials and methods

Chemicals

Standards for pyrethroids (cypermethrin, fenpropathrin, esfenvalerate, bifenthrin,

fenvalerate, deltamethrin, cyfluthrin, cyhalothrin, tau-fluvalinate, cyphenothrin, cis-

permethrin, flucythrinate and ethofenprox) were purchased from the TianjinInstitute for Environmental Protection (Tianjin, China). Carrier protein including

keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA)

were bought from BoAo Co., Shanghai, China. RPMI-1640 medium and foetal calf

serum were purchased from Sunshine Biotechnology Co. (Nanjing, China).

Chemicals for hapten modification including N-hydroxysuccinimide (NHS) and 1-

(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC) and N, N?-carbonyldiimida-

zole (CDI) were obtained from Sigma Chemical Co. (St. Louis, MO). Freund’s

adjuvant, 3,3?,5,5?-tetramethylbenzidine (TMB) and goat anti-mouse IgG (H�L)horseradish peroxidase(HRP) conjugate were also bought from Sigma Chemical Co.

All other chemicals and reagents were of analytical grade or those of higher grade

were obtained from East China Chemicals Co., Ltd., Wuxi, China.

Hapten synthesis and characterisation

Four haptens were synthesised. Hapten 1, (RS)-cyano-3-phenoxybenzyl-2, 2, 3, and 3-tetramethylcyclopropane-carboxylic acid (Figure 1b) were designed based on cypheno-

thrin. First, cyphenothrin (Figure 1a, 1.20 g, 3.2 mmol, mixed isomer) was suspended

in a mixed solution of tert-butyl alcohol (7.0 mL) and distilled water (17.0 mL). Then,

sodium periodate (47.06 mg, 0.22 mmol) and potassium permanganate (42.57 mg, 0.27

mmol) were added into the above solution, followed by the addition of sodium

carbonate (66.68 mg, 0.63 mmol) to adjust the pH of the reaction solution. After

stirring for 30 min at room temperature, the phase transfer catalyst was finally added

and the mixture was stirred at room temperature overnight. The reaction solution wasmonitored by thin-layer chromatography (TLC). Then, the final solution was acidified

to pH 2.0 with 6 N HCl and extracted three times with ethyl acetate. All organic phases

were combined and washed with 0.1 M Na2S2O5 solution. The harvested mixture was

dried over anhydrous MgSO4, and concentrated under reduced pressure to yield tan-

yellow oil. The crude oil was purified using a silica gel column (ethyl acetate: petroleum

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ether �1:5) to yield a light yellow oil (hapten 1, see Figure 1b). The product was

analysed by TLC and further confirmed using an all-digital nuclear magnetic resonance

spectrometer (NMR, AVANCE III, Bruker, Billerica, MA).

Hapten 2 (Figure 1c), 3-phenoxybenzoic acid (PBA), was purchased from J&K

Chemical Company (Tokyo, Japan). Hapten 3 (Figure 1d), 4-(cyano(3-phenoxyphe-

nyl)methoxy)-4-oxobutanoic acid, was synthesised referring to previous studies (Haoet al., 2009). Hapten 4 (Figure 1e) (N-(3-phenoxybenzoyl)-4-amino-L-phenylalanine)

was prepared by modifying the reported method (Shan et al., 1999).

Antigen synthesis

Haptens 1, 2 and 3 were conjugated with carrier protein including KLH, BSA and

OVA using activated ester methods (Kondo et al., 2012) and CDI methods (Shan,

Stoutamire, Wengatz, Gee, & Hammock, 1999). Hapten 4-containing amino group

was coupled to carrier protein by using the diazotisation method (Ahn et al., 2012).

All the resulting conjugates were characterised with a UV-vis spectrometer (Bokin

Instruments,Tsushima, Japan). The final products were divided into aliquots (0.2 mLper tube) and stored at �208C for further use.

Production of monoclonal antibodies

Immunisation and antiserum preparation

An immunisation programme was performed, modifying a reported format (Lin et al.,

2011). Eight-week-old female BALB/c mice were immunised with a mixture of antigen

O KMnO4

NaIO4

O COH

O

Hapten2

O

O

HN

COOHNH2

hapten4

CN

O

CO

O

CN

O

CO

CO

HO

a bHapten1cyphenothrin

(RS)-cyano-3-phenoxybenzyl-2,2,3,3-tetramethylcyclopropane-carboxylicacid

c

O

CN

O

O

O

HOd

Hapten3

3-phenoxybenzoicacid(PBA) 4-(cyano(3-phenoxyphenyl)methoxy-4-oxobutanoicacid

e

N-(3-phenoxybenzoyl)-4-amino-L-phenylalanine

Figure 1. Chemical structures for haptens.

Food and Agricultural Immunology 343

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solution (the conjugates of hapten1 and KLH) and Freund’s adjuvant. The dosage was

0.2 ml per mouse (containing 100 mg antigen). The booster inoculations were carried

out at 3-week intervals. Ten days after the last immunisation, antisera from mice were

tested using indirect competitive enzyme-linked immunosorbent assay (ci-ELISA).The mouse was sacrificed for cell fusion. The cycle of hybridoma selection referred to a

previous publication (Peng et al., 2012). The chosen cell line was expanded and

injected intraperitoneally into mice (106 cells /mouse). Ascites was collected and then

purified with caprylic acid and ammonium sulphate precipitation (Redwan, 2006).

Development of ci-ELISA for pyrethroids

The procedure of this ci-ELISA was similar to the description by Hao et al. (2009).

The performance of this assay was optimised to achieve the best sensitivity. The

combination of antibody and coating antigen was screened using a checker-board

titration system (ArunKumar, Basith, & Gomathinayagam, 2012). The effects of

ionic strength on assay performance were estimated by changing sodium chloride

content (0%, 0.5%, 0.8%, 1%, 2%, 4%, g/L) in assay buffer. In experiments toevaluate pH effects on assay sensitivity, four different pH values (5, 6, 7, 8) in assay

buffer were tested. Pyrethroids are lipophilic and the effects of co-solvents including

methanol, ethanol, acetone and dimethylformamide (DMF) on the ELISA

performance were determined using the aforementioned method. A standard

inhibition curve was obtained based on the optimised conditions and the developed

ELISA sensitivity was evaluated by the IC50 value. The cross-reactivity (CR%) was

determined using the reported formula (Sun, Liu, Kuang, & Xu, 2013):

CRð%Þ ¼ ðIC50 cypermethrin=IC50 related analoguesÞ � 100:

Sample detection

In this study, the local lake water (Li lake, Wuxi, China) was used to verify the

performance of the developed immunoassay. The water sample, which was confirmed

by GC-MS and showed without pyrethroid residues (less than the detection limit, 0.1

ng mL�1), was used for recovery tests.

Blank water samples were spiked with three representive pyrethroids at different

levels depending the chemicals (20, 40 and 100 ng mL�1 for cypermethrin, 50, 100

and 200 ng mL�1 for fenpropathrin and 250, 500 and 1000 ng mL�1 for

esfenvalerate). The water samples were simply diluted 10-fold using assay bufferbefore detetion with ELISA. Intra-day variation was determined based on 5

replicates and inter-day variation was measured on three consecutive days.

Results and discussion

Hapten characterisation and antigen preparation

Four haptens were synthetised for preparing diverse antigens in this text. Hapten 1

was a mimic of type II synthetic pyrethroids with three common structures containing

a phenoxybenzyl group, a dimethylcyclopropane, and an a-cyano group and then was

chosen as an immune hapten. In order to improve the sensitivity of ELISA method,

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hapten 2, hapten 3 and hapten 4 were produced and used as coating haptens. Based on

TLC analysis, the rate of flow (Rf) of hapten1 was calculated as 0.36 using a mobile

phrase of mixed solvents (ethyl acetate: petroleum ether: acetic acid �30:69.9:0.1, v/

v). The NMR information indicated that hapten1 had been synthesised successfully.

The NMR analysis was based on chemical shift values in parts per million (ppm) with

tetramethylsilane as internal standard: 1H NMR (400 MHz, DMSO-d6) d 7.52 (t,

J �8.0 Hz, 1H, ArH), 7.44 (t, J�8.0 Hz, 2H, ArH), 7.37 (t, J �7.2 Hz, 1H, ArH),

7.22 (m, 2H, ArH), 7.12-7.06 (m, 3H, ArH), 6.71 (d, J �13.2 Hz, 1H, CHCN), 2.12

(m, 2H, CH), 1.24 (d, J �9.2 Hz, 3H, CH3), 1.22 (d, J �10.0 Hz, 3H, CH3).

All haptens showed major UV absorption peaks at 230 and 275 nm while the main

UV absorption peaks of carrier protein OVA and BSA were 210 and 280 nm. Besides

the main UV peak at 210 and 280 nm, KLH has another specific UV absorption peak

at 350 nm. For the prepared conjugates, it is much easy to identify that the products

have characteristic UV peaks containing carrier protein and haptens. In addition,

obvious UV peak shift (5�10 nm) could be found for synthesised antigens.

Establishment of ELISA for pyrethroids

Eight coating antigens, OVA and BSA conjugates with four haptens, respectively,

were used to optimise the ELISA method. Optimum concentrations of antibody and

coating antigen in ELISA system were selected based on the checkerboard

Figure 2. Effects of ionic strength (a), pH (b) and methanol content (c) in assay buffer on the

performance of ELISA and the standard inhibition curve under the optimised conditions (d).

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experiment. The monoclonal antibody showed lower affinity to coating antigens of

hapten 3 and hapten 2 (the maximum OD value 0.5 and 0.7, respectively). A lowest

IC50 for cypermethrin was obtained using the combination of coating antigen

(hapten 4-BSA, 0.5 mg mL�1) and monoclonal antibody 3E9 (0.04 mg mL�1).Ionic strength in assay system was changed with different NaCl content in assay

buffer (0.01 M phosphate buffer, PB). Results (Figure 2a) demonstrated that NaCl

content in the assay buffer affected IC50 value to a large extent. To maximise the OD

value while minimising the IC50, 1% (m/v) NaCl was optimal and used in subsequent

experiments. Results (as can be seen from Figure 2b) showed that the changes of pH

values from 5 to 8 had little effects on the ELISA system, which demonstrated the

rigidity and stability of this ELISA system. A common assay buffer at pH 7.2 was

chosen for following experiments.Co-solvent was necessary to solubilise pyrethroids in water and to extract

pyrethroids from matrix. Four water-miscible solvents including DMF, ethanol,

acetone and methanol were tested in our experiments. The antibody seemed sensitive

to the content of DMF and ethanol in assay buffer. In addition, acetone as an assistant

solvent resulted in higher background value of ELISA detection. The possible co-

extracts from water samples may affect the recognition between antibody and antigen.

Comparatively speaking, the immunoassay showed good tolerance to methanol

content in assay buffer (Figure 2c). Twenty percent methanol in 0.01 M phosphate-buffered saline was used to dilute water samples in this ELISA. Under the optimised

conditions, the IC50 value for each pyrethroid was calculated based on the inhibition

curves (Figure 2d) and showed that IC50 value was cypermethrin, 1.790.76 ng mL�1,

fenpropathrin, 14.091.68 ng mL�1, and esfenvalerate, 45.894.07 ng mL�1,

respectively.

Cross-reactivity

More than 20 pyrethroids were tested for cross-reaction of this immunoassay. The

monoclonal antibody 3E9 shows best specificity to cypermethrin and affinity to

fenpropathrin, esfenvalerate, bifenthrin, deltamethrin and fenvalerate in different

degrees (Table 1). The cross-reactivity results indicated that antibody 3E9 preferred

phenoxy moiety to cyclopropane part in recognisation of pyrethroids.

Recovery studies

The recovery for spiked lake water samples was calculated based on the standard

curve in Figure 2d and the data are shown in Table 2. The recoveries for the three

pyrethroids ranged from 76 to 118% with the intra-day coefficients of variation (CV)

4.31�8.60% and inter-day CV value 8.57�14.10%. Synthetic pyrethroids were highly

toxic for some aquatic species, such as fish, shrimp, etc. (Zhang et al., 2012). The

proposed immunoassay provides a direct detection for pyrethroids in water with a

simple dilution treatment of samples.

Conclusions

A monoclonal antibody was developed with good recognition to cypermethrin and

broad affinity to other five pyrethroids. Various parameters including pH, ionic

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Table 1. Cross-reactivity in ELISA.

Chemicals Structure IC50 ng mL�1 (n�5) CR (%)

Cypermethrin 1.6690.76 100

Fenpropathrin 14.0391.68 11.83

Esfenvalerate 45.7694.07 3.63

Bifenthrin 191.8911.2 0.87

Deltamethrin 199.6910.75 0.83

Fenvalerate 298.5915.08 0.61

Hapten1 313.5915.96 0.53

Hapten 2 482.6925.12 0.35

Hapten 4 74.298.75 2.24

Hapten3 �1000 B0.2

Cyhalothrin �1000 B0.2

Cis-permethrin �1000 B0.2

Fluvalinate �1000 B0.2

Cyfluthrin �1000 B0.2

Cyphenothrin �10000 B0.02

Flucythrinate �10000 B0.02

Ethofenprox �10000 B0.02

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strength and co-solvents were tested to improve the performance of immunoassay.

The proposed ELISA method was successfully applied to detect water samples with

satisfactory recovery rate at different spiked levels. The research provided a cost-

effective immunoassay in pyrethroid detection, and this developed method could be

used to monitor pyrethroid residues in agricultural production.

Acknowledgements

This work is financially supported by the National Natural Science Foundation of China(21071066, 91027038, 21101079, 21175034), the Key Programs from MOST (2012BAC01B07,2012BAD29B05, 2012AA06A303, 2012BAD29B04, 2011BAK10B07, 2011BAK10B05, 2011BAK10B01, 2010AA06Z302, 2010DFB3047, 2011ZX08012-001, 2012BAK17B10, 2012BAK08B01, 2012YQ090194) and grants from Jiangsu Province, MOF and MOE (NCET-12-0879,BE2011626, 201210036, 201310135, 311002).

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Coefficients of variation

(CV,%)

Analyte

Spiked level

(ng mL�1)

Mean recovery

(%)

Intra-day

(n�5)

Inter-day

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100 92.7 7.15 8.96

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100 90.7 5.71 9.62

200 89.3 4.05 14.10

Esfenvalerate 250 111.3 5.49 10.03

500 99.3 7.50 8.57

1000 90.7 8.60 10.47

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