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Enzyme-linked immunosorbentassay–based two different polyclonalantibodies for the detection ofcypermethrin with phenoxybenzenemultiresidueXinlin Weia, Yaru Zhaoa, Baoqin Wanga & Yuanfeng Wanga

a Institute of Food Engineering, College of Life & EnvironmentScience, Shanghai Normal University, Shanghai, PR ChinaPublished online: 02 Jul 2013.

To cite this article: Xinlin Wei, Yaru Zhao, Baoqin Wang & Yuanfeng Wang (2014) Enzyme-linkedimmunosorbent assay–based two different polyclonal antibodies for the detection of cypermethrinwith phenoxybenzene multiresidue, Food and Agricultural Immunology, 25:3, 364-374, DOI:10.1080/09540105.2013.805732

To link to this article: http://dx.doi.org/10.1080/09540105.2013.805732

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Enzyme-linked immunosorbent assay�based two different polyclonalantibodies for the detection of cypermethrin with phenoxybenzenemultiresidue

Xinlin Wei, Yaru Zhao, Baoqin Wang and Yuanfeng Wang*

Institute of Food Engineering, College of Life & Environment Science, Shanghai NormalUniversity, Shanghai, PR China

(Received 23 March 2013; final version received 13 May 2013)

Two kinds of new immunogens and coating antigens had been designed andsynthesised by methods of lively ester (DCC) and carbodiimide (EDC), respectively.Preliminary immunosorbent assays indicated that the two target conjugatespresented good inhibition ratio against the haptens of cypermethrin in vivo. Theresults showed that the best efficient titres of anti-cypermethrin rabbit serum inmethods of DCC and EDC were 1:32000 and 1:64000, the IC50 of them were18.907mg/L and 16.877mg/L against the haptens of cypermethrin, respectively. Thedata indicated that the anti-cypermethrin antibodies in method of EDC had highersensitivity and higher selectivity than the anti-cypermethrin antibodies in methodof DCC. Thus, we can synthesise antigens and anti-bodies with method of EDC.The antibodies of high quality can be effectively used in the field of the rapiddetection of pyrethroid pesticide residues in the research of the reagent strips.

Keywords: cypermethrin; DCC; EDC; haptens; enzyme-linked immunosorbentassay

1. Introduction

As a group of bionic insecticides with relatively high potent and low mammalian

toxicity, pyrethroids are being widely used in forestry, horticulture, agriculture

situations and public health throughout the world (Leahey, 1985; Miadokova et al.,

1992). Cypermethrin is a stable pyrethroid with insecticidal activity. Its greater

photostability to degradation than other pyrethroids has made it attractive for uses

requiring longer residual activity. Cypermethrin has been reported as surface water

contaminants and impacts the environment, leading to effects on ecosystem health

(Moore & Waring, 2001). Many of the toxicological studies on pyrethroids focused

on nontarget vertebrates and mammalian animals (Coats et al., 1989; Haya, 1989;

Schimmel, Garnas, Patrick, & Moore, 1983). Thus, a sensitive and selective method

for monitoring residue levels of cypermethrin is needed, particularly in public health.

Current analytical methods for cypermethrin involve spectrophotography, gas

chromatography-electron capture (GC-EC), thin layer chromatography (TLC) or high-

performance liquid chromatography-mass spectrometry (HPLCMS) (Akhtar, 1982;

Ding, Bao, & Zheng, 2000; Esteve-Turrillas, Pastor, & de la Guardia, 2005; Ferrer

*Corresponding author. Email: foodlab2010@gmail.comXinlin Wei and Yaru Zhao have contributed equally for this study.

Food and Agricultural Immunology, 2014

Vol. 25, No. 3, 364�374, http://dx.doi.org/10.1080/09540105.2013.805732

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et al., 2005; Li, Huang, Dai, & Zhang, 2002). These methods are relatively expensive

and time consuming, and are not significantly suitable for large numbers of samples.

An immunoassay, such as enzyme linked immunosorbent assay (ELISA), would

provide a sensitive and selective method for the detection of this pyrethroid at tracelevels (Hammock & Mumma, 1980; Hammock et al., 1989; Shan, Stoutamire,

Wengatz, Gee, & Hammock, 1999; Shan, Wengatz, Stoutamire, Gee, & Hammock,

1999; Shan et al., 2000; Wing & Hammock, 1979; Wing, Hammock, & Wustner, 1978).

At present, in the method of ELISA, the quality of antiserum determines the

detective effect against cypermethrin. The quality evaluation of antiserum’s detection of

pesticide residue is composed of three indicators: antiserum titre, half inhibition rate and

cross-reactivity (CR) rate, which are evaluated by serum antibody content, antiserum on

target detection of small molecule detection sensitivity and specificity (Lei et al., 2010;Suarez-Pantaleon, Mercader, Agullo, Abad-Somovilla, & Abad-Fuentes, 2010).

To raise the selectivity of antibodies against cypermethrin, the cypermethrin

molecule must be modified to attach an arm to carry protein. Two haptens with

phenoxybenzene multiresidue were designed and synthesised utilizing two different

types of synthesis. The structures of the two target haptens were confirmed by 1H

NMR, IR, MS and elemental analysis. Serum antibody had been made by two

different anti-cypermethrin antibodies which been prepared by two different

immunizing New Zealand white rabbits. Previous studies of pyrethroid pesticideonly utilised one method to judge the quality of serum antibody. The methods of

preparation of cypermethrin antibody in this article can help the preparation of

cypermethrin in the prospective research.

2. Materials and methods

2.1. Materials

Cypermethrin, Fenvalerate, Cyhalothrin and Deltamethrin were purchased from

Nanjing ronch. Sodium azide was purchased from Aladdin. Bovine serum albumin

(BSA), Albumin egg (OVA), TMB, Enzyme-labelled goat anti-rabbit, Tween-20 and

Tris were purchased from Solarbio. Freund’s complete adjuvant and Freund’sincomplete adjuvant were purchased from Sigma. (NH4)2SO4, Na2HPO4 12H2O,

NaH2PO4 2H2O, NaCl, Hydrochloric acid and Methanol were purchased from Scrc.

2.2. Equipment

Magnetic stirring instrument was purchased from IKA. Full wavelength UV

spectrophotometer was purchased from Pgeneral. Pipettes were purchased from

Eppendorf. Microplate reader and Infrared spectrometer were purchased from

Thermo. Water treatment systems were purchased from Siemens. Analytical balance

was purchased from Sartorius.

3. Methods

3.1. Antigen synthesis

Ethylenediamine and 1,3-propylene amide as connecting arm was added to the

cypermethrin hapten by methods of lively ester (DCC) and carbodiimide (EDC),

respectively (Lee, McAdam, & Skerritt, 1998). Specific synthetic steps are as follows

after Figure 1:

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(9)-1-Carboxy-(3-phenoxy phenyl) methanol

To a flask containing cypermethrin (5.6g, 0.11mol) was added 100mL of 1 M NaOH

in 60% tetrahydrofuran (THF), and the mixture was refluxed for 3 days. The

resulting solution was acidified using concentrated HCl (caution) and extracted three

times with ethyl acetate. The combined organic layers were then washed by 0.1M

HCl and acetone, dried over MgSO4 and concentrated under evaporation to yield a

light yellow solid.

3.1.1. Method of DCC

(9)N-(2-aminoethyl)-2-hydroxy-2-(3-phenoxyphenyl)acetamide

The compound1 was added in dimethylformamide and cooled in an ice bath for 30

min, and it was treated with NHS, DCC, ethylene diamine and after recrystallisation

from ethyl to yield a light yellow solid: yield:60.7%; light yellow solid, mp 178�1808C; 1H NMR (400 MHz, CDCl3) d: 7.30�7.72 (m, 3H), 6.90�7.29 (m, 5H), 6.84

(m, J �7.6 Hz, 2H), 5.58 (s, 1H), 3.11 (d, J �14.6 Hz, 2H), 2.89 (d, J �14.8 Hz, 2H),

OHO

CNHCl O

HO

COOH

N

O

O

HO

DCC

BSA

OHO

CO

RT

RT 3h

3h 4°C 6h

1

2

1

1

N

O

O

HO

EDC RT 3h

BSA

4°C 3h

3

HN

HNBSA

OHO

CO

HN

NH2H2N

NH2

H2N NH2

OHO

CO

HN NH2

OHO

CO

HN

HN

BSA

4

5

OO

CNOClCl

NaOH

OH

OClCl

OHO

CN

72h70°C

1

Figure 1. Scheme for hapten synthesis.

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2.08 (m, 3H); ESI-MS ([M�H]�) m/z: 287.14. Anal. calcd for C16H18N2O3: C%

67.12, H% 6.34, N% 9.78; found C% 67.19, H% 6.37, N% 9.72.

Dissolve BSA in the phosphate buffer, and the DMF was added in the mixture

and then refluxed for 6 hours at 48C. Finally, the mixture was taken into dialysis bagat 48C for 72 hours. Then the mixture was conserved at �208C.

Coating antigen synthesis: The method of coating antigen synthesis was the same

as the method of antigen synthesis, where the OVA was used instead of the BSA.

3.1.2. Method of EDC

(9)N-(3-aminopropyl)-2-hydroxy-2-(3-phenoxyphenyl)acetamide

The compound1 (0.572g, 0.002mol) was added in dimethylformamide (2ml), Na2SO4

(0.3g) and cooled in an ice bath for 30 min, then it was treated with NHS, EDC, 3-

dimethyl aminopropyl amine, and after recrystallisation from ethyl to yield a light

yellow solid: yield: 62.4%; light yellow solid, mp 186�1888C; 1H NMR (400 MHz,

CDCl3) d: 7.30�7.72 (m, 3H), 6.90�7.29 (m, 5H), 6.84 (m, J �7.6 Hz, 2H), 5.58 (s,

1H), 3.11 (d, J �14.6 Hz, 2H), 2.89 (d, J �14.8Hz, 2H), 2.08 (m, 3H), 1.80 (d, J �14.8Hz, 2H), ESI-MS ([M�H]�) m/z: 301.16. Anal. calcd for C17H20N2O3: C%

67.98, H% 6.71, N% 9.33; found C% 68.01, H% 6.76, N% 9.31.

Dissolve BSA (0.2g) in the phosphate buffer (0.02mol/L 15mL), and the front

mixture was added in the latter mixture and then refluxed for 3 hours at 48C. Finally,

the mixture was taken into dialysis bag at 48C for 72 hours. Then the mixture was

conserved at �208C.

Coating antigen synthesis:The method of coating antigen synthesis was the same

as the method of antigen synthesis, where the OVA was used to instead of the BSA.

3.2. Immunisation and antiserum preparation

Cypermethrin antisera were obtained following the protocol described previously

(Shan, Wengatz, et al., 1999). New Zealand white rabbits were immunisedintradermally with each immunogen hapten 2-BSA (rabbit #1, and rabbit #2),

3-BSA (rabbit #3and rabbit #4). One month after an initial immunisation with 1 mg/

mL of the immunogen protein emulsified with Freund’s complete adjuvant (1:1, v:v),

further injections of 1mg/mL of the immunogen emulsified with Freund’s incomplete

adjuvant were given(1:1, v:v). Booster injections were given at six-week intervals. The

rabbits were bled 7 days after each boost. The serum was isolated by centrifugation

for 10 min at 48C and stored at �208C. The results of antibody characterisation were

obtained from sera of terminal bleed after seven boosters.

4. Screening and selection of antisera

4.1. Enzyme-linked immunosorbent assay indirectly

The method was performed as previously described by Shan, Stoutamire, et al. (1999;Kong, Zhang, Zhang, Gee, & Li, 2010; Lei et al., 2010). Microplates were coated

overnight at 48C with 100mL per well of the appropriate coating antigen

concentration in 0.1 M carbonate-bicarbonate buffer (pH �9.6). After the plate

had been washed with washing solution (0.05% Tween-20 in 0.01M PBS (pH �7.4)),

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the surface of the wells was blocked with 200mL of 0.5% OVA in 0.01M PBS (8g

NaCl, 2.9g Na2HPO4 �12H2O, 0.24g KH2PO4, 0.2g KCl) by incubation for 2h at room

temperature to minimise the non-specific binding in the plate. After another washing

step, 100mL of antiserum per well serial diluted in PBS (for titration experiments) wasadded and incubated for 1h at room temperature. The standard analyte concentra-

tions ranged from 1mg/mL to 9.8�10�4mg/mL. Following a washing step, goat anti-

rabbit IgG-HRP conjugate (diluted in 1:2000 in PBS with 0.05% Tween-20, 100mL per

well) was added and incubated for 1h at room temperature. The plates were washed

for five times (three times in PBST and two times in distilled water), and 100mL per

well of TMB was added. The colour development stopped after 20min with 50mL per

well of 2M H2SO4. The absorbance was measured using a dual wavelength mode at

450nm. Then the titres were obtained by optimal dilution factors. The standard ofselecting the titre of antiserum was the OD values equal 1.0.

4.2. Competing enzyme-linked immunosorbent assay

The method was performed as previously described by (Lei et al., 2010; Shan,

Stoutamire, et al., 1999; Suarez-Pantaleon et al., 2010). Microplates were coated

overnight at 48C with 100mL per well of the appropriate coating antigen

concentration in 0.1M carbonate-bicarbonate buffer (pH �9.6).After the plate had

been washed with washing solution (0.05% Tween-20 in 0.01M PBS (pH �7.4)), thesurface of the wells was blocked with 200mL of 0.5% OVA in 0.01M PBS (8g NaCl,

2.9g Na2HPO4 �12H2O, 0.24g KH2PO4, 0.2g KCl) by incubation for 2h at room

temperature to minimise the non-specific binding in the plate. After next washing

step, 100mL per well of antiserum diluted in PBS (for titration experiments) followed

by the titre of indirectly ELISA and 100mL per well of analyte solution was added

and incubated for 1.0h at room temperature. The standard analyte concentrations

ranged from 1mg/mL to 9.8�10�4mg/mL. Following a washing step, goat anti-

rabbit IgG-HRP conjugate (diluted in 1:2000 in PBS with 0.05% Tween-20, 100mLper well) was added and incubated for 1h at room temperature. The plates were

washed for five times (three times in PBST and two times in distilled water), and

100mL per well of TMB was added. The colour development stopped after 20 min

with 50mL per well of 2M H2SO4. The absorbance was measured using a dual

wavelength mode at 450nm. Standard curves were obtained by plotting absorbance

against the logarithm of analyte concentration, which were fitted to a four parameter

logistic equation:

y ¼ ðA�DÞ=½1þ ðx=CÞB� þD

where A is the maximum absorbance at no analyte present; B is the curve slope at the

inflection point; C is the concentration of analyte giving 50% inhibition (IC50); D is

the minimum absorbance at infinite concentration.

4.3. Cross-reactivity

The optimised assays were applied to CR studies by using the standard solution of

the analyte and other structurally related compounds. The CR was quantified by

comparing the IC50 of the cypermethrin and another compound.

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5. Results

5.1. Hapten synthesis

Two different haptens were synthesised with different linking groups to reduce the

affinity of the antibodies for the spacer arm in immunoassays. The haptens were

synthesised according to Figure 1. The structures of the two target haptens were

confirmed by 1H NMR, MS and elemental analysis. The synthetic routes parallel

tothose described for the synthesis of cypermethrin haptens in a previous publication(Lee et al., 1998). The immunogens and coating antigens were detected by UV assays.

Cypermethrin is a pyrethroid insecticide containing three chiral centres. Hence,

the haptens were synthesised as mixtures of two diastereoisomers racemic at the

cyanohydrin centre to allow detection of both of two isomers. These reaction

conditions used would possibly lead to isomerisation during these syntheses.

Therefore, syntheses were carried out using racemic mixtures without identification

or separation of each stereoisomer.

5.1.1. UV absorbance spectrum of cypermethrin

The immunogens and coating antigens were detected by UV assays (Li, Qiu, &

Wang, 2002; Wang, Dong, & Zou, 2006).

Figures 2�5 indicated that immunogens and coating antigens have been obtained,

respectively.

5.2. Screening and selection of antisera

5.2.1. The results of enzyme-linked immunosorbent assay indirectly

In Figure 6, the result showed that the titre of immunogen of cypermethrin in rabbits’

serum increase rapidly after the fourth immunisation, and the titre rose not

significant after the fifth immunisation. Then take the impact immunisation by ear

vein in the seventh immunisation booster. The Figure 6 showed the titres of rabbit#3

and rabbit#4 (method of DCC) were higher than rabbit#1 and rabbits#2 (method of

EDC). The most efficient titres of anti-cypermethrin rabbit serum in methods of

DCC and EDC were 1:32000 and 1:64000. So the rabbit#1 and rabbit#3 wereselected as the research of evaluation system.

3203002802600.0

0.5

1.0

(a) BSA (b) Hapten1 (c) Hapten1-BSA

(c)

(b)

(a)

OD

Wavelength (nm)

Figure 2. The immunogen of cypermethrin in DCC.

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5.2.2. Competing enzyme-linked immunosorbent assay

In the Figure 7, the results showed that the IC50 of rabbit#1 and rabbit#3 were

18.907mg/L and 16.877mg/L against the haptens of cypermethrin, respectively. And

the lower detectabilities were 18.222mg/L and 16.323mg/L. The detection ranges of

them were 18.222mg/L to 22.499mg/L and 16.323mg/L to 20.654mg/L.

The antisera of terminal bleed from four rabbits were screened against two

different coating antigens using a two-dimensional titration method with the coated

antigen format. Figures 6 and 7 were the competition curve about the rabbit#1 and

rabbit#3 anti-cypermethrin serums. The IC50 of them were 18.9072mg/L and

16.8774mg/L against cypermethrin respectively. The detectability of the No.3 rabbit

serum is higher than the No.1 rabbit serum.

5.3. Cross-reactivities

The antibodies were made by combining the protein BSA with the phenoxybenzyl

group. It was expected that the ability of the antibody to distinguish among

cypermethrins would be more than other pyrethroid molecules.

0.0

0.5

(a) OVA (b) Hapten1 (c) Hapten1-OVA

(c)

(b)

(a)O

D

320300280260Wavelength (nm)

Figure 3. The coating antigen of cypermethrin in DCC.

0.0

0.5

1.0

(a)

(a) BSA (b) Hapten2 (c) Hapten2-BSA

(c)

(b)

OD

320300280260Wavelength (nm)

Figure 4. The immunogen of cypermethrin in EDC.

370 X. Wei et al.

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3 4 5 6 70

5000

10,000

15,000

20,000

25,000

30,000

35,000

40,000

45,000

50,000

55,000

60,000

65,000

Tite

r

Times

rabbit#1 rabbit#2 rabbit#3 rabbit#4

Figure 6. The titre of immunogen of cypermethrin.

5 10 15 20 25 300.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9(a) rabbits #1(b) rabbits #3

(b)

(a)

Pre

sent

of i

nhib

itory

con

cent

ratio

n

Concentration(µg/L)

Figure 7. Competitive inhibitory curve of cypermethrin by ELISA.

0.0

0.5

(a)

(a) OVA (b) Hapten2 (c) Hapten2-OVA

(c)

(b)

OD

320300280260Wavelength (nm)

Figure 5. The coating antigen of cypermethrin in EDC.

Food and Agricultural Immunology 371

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The data in Table 1 indicated that the rabbit#3 showed higher selectivity than

rabbit#1. It is clearly showed that chlorine in ethenyl and a cyclopropane ring was

important in increasing the selectivity.

6. Discussion

Two kinds of new immunogens and coating antigens had been designed and synthesisedby methods of lively ester (DCC) and carbodiimide (EDC), respectively. Immunosor-

bent assays indicated that the most efficient titres of anti-cypermethrin rabbit serum in

methods of DCC and EDC were 1:32000 and 1:64000, the IC50 for cypermethrin

were 18.907mg/L and 16.877mg/L and the lower detectabilities were 18.222mg/L and

16.323mg/L. The detection ranges were 18.222mg/L to 22.499mg/L and 16.323mg/L to

20.654mg/L, respectively. The antibodies reported here were developed from a novel

cypermethrin hapten and resulted in an assay that is 2 times more sensitive than

previously reported for unisomerised cypermethrin. The data indicated that the anti-cypermethrin antibodies in method of EDC have higher sensitivity and higher

selectivity than the anti-cypermethrin antibodies in method of DCC. Thus, method

of EDC can be utilised to synthesise antigens and anti-bodies in the next research.

The results with the titre, the IC50 and the cross-reactivities showed that the anti-

cypermethrin antibodies in method of EDC have higher sensitivity and higher

selectivity than the anti-cypermethrin antibodies in method of DCC. As shown in

Figure 7, conjugate 5 exhibited better competitive inhibitory curve than conjugate 4.

Therefore, these results suggest that the inhibition ratios of conjugates 4 and 5 werecontrolled by the length of side chain and the size of the hapten molecule. The

change of length of side chains and the sizes of the hapten molecule contributed to

obtain immunogens and coating antigens with better quality, perhaps.

Acknowledgements

We gratefully acknowledge the financial support of the National Natural Science Foundationof China (No. 81072308), Shanghai Biomedicine Key Program (No. 10391901700,No. 08391911100), the Program of Food Safety and Nutrition Team of Shanghai Normal

Table 1. Cross-reactivities.

IC50(g/L) Cross-reactivities (%)

Substance Structural DCC EDC DCC EDC

Cypermethrin OO

CNOClCl 18.907 16.877 100 100

Fenvalerate OO

CNO

Cl 22.331 19.343 84.67 87.25

Cyhalothrin OO

CNOCl

F3C 22.409 19.101 84.67 88.36

Deltamethrin OO

CNOBr

Br22.854 19.259 82.73 87.63

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University (DXL123), Shanghai Yangtze River Delta Science Joint Efforts Program 255(12495810600,11495810500), Development Center of Plant Germplasm Resources of Shang-hai (B-6010-11-001), and local colleges and universities’ capacity building program.

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