Analytica Chimica Acta 571 (2006) 79–85

Comparison between conventional indirect competitiveenzyme-linked immunosorbent assay (icELISA) and

simplified icELISA for small molecules

Jing Zhao a, Gang Li a, Guo-Xiang Yi a, Bao-Min Wang a,∗, Ai-Xing Deng a,Tie-Gui Nan a, Zhao-Hu Li a, Qing X. Li b

a College of Agronomy and Biotechnology, China Agricultural University, Beijing 100094, Chinab Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, HI 96822, USA

Received 17 November 2005; received in revised form 21 February 2006; accepted 24 April 2006Available online 29 April 2006

Abstract

A simplified indirect competitive enzyme-linked immunosorbent assay (icELISA) for small molecules was established by modifying the proce-dagaiwcbb©

K

1

mdc(mitiait

0d

ure of conventional icELISA. The key change was that the analyte, antibody, and enzyme-labeled second antibody in the simplified icELISA weredded in one step, whereas in conventional icELISA these reagents were added in two separate steps. Three small chemicals, namely zeatin riboside,lycyrrhetinic acid, and chlorimuron-ethyl, were used to verify the new assay format and compare the results obtained from conventional icELISAnd simplified icELISA. The results indicated that, under optimized conditions, the new assay offered several advantages over the conventionalcELISA, which are simpler, less time consuming and higher sensitive although it requires more amount of reagents. The assay sensitivity (IC50)as improved for 1.2–1.4-fold. Four licorice roots samples were analyzed by conventional icELISA and simplified icELISA, as well as liquid

hromatography (LC). There was no significant difference among the content obtained from the three methods for each sample. The correlationetween data obtained from conventional icELISA and simplified icELISA analyses was 0.9888. The results suggest that the simplified icELISAe useful for high throughput screening of small molecules.

2006 Elsevier B.V. All rights reserved.

eywords: icELISA; Small molecules; Chlorimuron-ethyl; Glycyrrhetinic acid; Zeatin riboside

. Introduction

Immunoassays were widely used for the analysis of smallolecules such as pesticides [1–8], plant hormones [9–11], tra-

itional Chinese medicines [12,13] and other environmentalhemicals [14–16]. The enzyme-linked immunosorbent assayELISA) was one of the most commonly used immunochemicalethods. For example, the fraction of ELISAs in immunochem-

cal methods for determining pesticides was about 90% [1]. Theechnique presently favored is competitive ELISAs. Compet-tive ELISAs include indirect competitive ELISA (icELISA)nd direct competitive ELISA (dcELISA) [1]. The icELISAs an immobilized antigen assay, which is based on competi-ion between the immobilized antigen and an unknown amount

∗ Corresponding author. Tel.: +86 10 6273 1305; fax: +86 10 6273 2567.E-mail address: wbaomin@263.net (B.-M. Wang).

of analyte for a small fixed amount of antibody. The antibodybound to the immobilized antigen is quantitated by the activityof an enzyme-labeled second antibody. In general, the procedureof conventional icELISA (Fig. 1a) includes coating microtiterplates with a coating antigen, blocking, a competitive reactionbetween free analyte and the hapten–protein conjugate for theantibody, the addition of the enzyme-labeled secondary anti-body, and the color reaction. Between each of these steps, thereis a washing step. The dcELISA (Fig. 1b) is an immobilized spe-cific antibody assay, which is based on competition between aconstant amount of the hapten–enzyme and the unknown analytefor a limited amount of antibody. The degree of color developedis inversely proportional to the amount of analyte being inves-tigated. The dcELISA procedure includes coating microtiterplates with a specific antibody, blocking, a competitive reac-tion between the free analyte and the enzyme conjugate for thecoating antibody, and a final color reaction. Between each ofthese steps, there is also a washing step. Both assay formats have

003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2006.04.060

80 J. Zhao et al. / Analytica Chimica Acta 571 (2006) 79–85

Fig. 1. Schematic presentations of (a) conventional icELISA, (b) dcELISA, and (c) simplified icELISA.

J. Zhao et al. / Analytica Chimica Acta 571 (2006) 79–85 81

advantages and disadvantages. In the case of the dcELISA, theadvantage is that it is a simple and time-saving procedure witha better detection limit [17]. The disadvantages of the dcELISAare as follows: (i) the synthesis of the enzyme conjugate willdecrease the activity of enzyme; (ii) there is a possible effect ofthe sample matrix on enzyme activity; (iii) the plate coated withthe antibody cannot be stored for a long time without specialtreatment at 4 ◦C; (iv) the amount of antibody used for coatingin dcELISA is more than that used in icELISA. However, it mustbe noted that all the problems have been solved and most com-mercial enzyme immunoassay kits for small molecules, suchas �-adrenoceptor, antibiotics, and natural toxins, are based onthis format. The main reason is that it is more convenient andsensitive than icELISA to screen the residues in animal tissuesand vegetables. The advantages of icELISA include (i) thereis no interference from the sample matrix on enzyme activitybecause the enzyme-labeled second antibody is pipetted to theplate in a separate step; (ii) the antigen-coated plate can be storedat 4 ◦C for a longer time than a plate coated with antibodies,even without special treatment; (iii) commercial enzyme-labeledsecond antibodies can be easily obtained. However, there arevery few commercially available enzyme immunoassay kits forsmall molecules based on the icELISA. The main reason is thaticELISA has more complicated procedures and a lower detec-tion limit than the dcELISA [17].

This study is to modify the conventional icELISA for a sim-psdpspab

2

I

2

(gh7CRw

2

pwm

2.3. Buffers and solutions

The following solutions were used: (i) a coating bufferconsisting of 0.05 M carbonate buffer, pH 9.6; (ii) phosphate-buffered saline (PBS), composed of 0.1 M phosphate buffercontaining 0.9% NaCl, pH 7.5; (iii) PBS with 0.1% (v/v) Tween(PBST); (iv) PBST contain 0.5% (w/v) gelatin (PBSTG); (v)citrate-phosphate buffer, consisting of 0.01 M citric acid mono-hydrate and 0.03 M Na2HPO4, pH 5.5; (vi) a substrate solu-tion, prepared by the addition of 4 �L of 30% H2O2 to 10 mLcitrate–phosphate buffer containing 2 mg mL−1 OPD; (vii) astop buffer, consisting of 2 M H2SO4.

2.4. Preparation of immunogen and coating antigen

2.4.1. Preparation of zeatin riboside (ZR) immunogen andcoating antigen [18]

ZR was covalently attached to BSA and OVA, respectively.Briefly, 1 mL of fresh 0.01 M NaIO4 water solution was addeddropwise to a methanol solution (2 mL) of ZR (2 mg) and stirredin the dark at room temperature for 20 min. The excess periodatewas destroyed by adding ethylene glycol (0.1 M, 60 �L). Themixture was stirred in the dark for 10 min at room temperatureand added to BSA solution (10 mg BSA or OVA in 2 mL ofdistilled water, and the pH was 9.3 adjusted with 5% K2CO3solution). After the reaction was carried out at room temperatureisKAfw

2a

atmnotts

2a

0lsHrwwT(

lified format (Fig. 1c). This new procedure involves fewerteps than the conventional icELISA, which is the same as thecELISA. Compared with the conventional icELISA, the sim-lified icELISA saves at least 0.5 h, and the sensitivity increasesignificantly over the conventional icELISA. However, the sim-lified icELISA has its inevitable disadvantages such as moremount of reagents (antibody and enzyme-linked second anti-ody) required than that used in conventional icELISA.

. Materials and methods

All data were analyzed using the SAS software program (SASnstitute, Cary, NC, USA).

.1. Reagents

Goat anti-rabbit IgG conjugated with horseradish peroxidaseIgG-HRP), o-phenylenediamine (OPD), zeatin riboside (ZR),lycyrrhetinic acid (GA), dicyclohexylcarbodiimide (DCC), N-ydroxysuccinimide (NHS), and 1,8-diazabicyclo[5.4.0] undec--ene (DBU) were purchased from Sigma (St Louis, MO, USA).hlorimuron-ethyl (CE) was obtained from Beijing Chemicaleagents Company (Beijing, China). All other chemicals usedere of analytical grade.

.2. Apparatus

The apparatus used in the present study included 96-wellolystyrene microtiter plates (Costar, USA), an automated plateasher (Wellwash 4 MK2, Thermo, Vantaa, Finland), and aicroplate reader (Multiskan MK3, Thermo, Vantaa, Finland).

n the dark for 1 h at pH 9.3, the solution was cooled to 4 ◦C. Theolution was incubated for 30 min after addition of 0.85 mg ofBH4 and for another 30 min with another 0.85 mg of KBH4.fter adjusting to pH 6.5 with 0.1 M HCl, the solution was stirred

or 1 h. The conjugate was dialysed exhaustively against distilledater and stored at −20 ◦C after lyophilization.

.4.2. Preparation of glycyrrhetinic acid (GA) immunogennd coating antigen

GA-BSA and GA-OVA conjugates were prepared via thective ester method. DCC (150 mg) was added to a stirring mix-ure of GA (80 mg) and NHS (40 mg) in 3 mL of DMF. The

ixture was stirred for 4 h at 4 ◦C and centrifuged. The super-atant was added dropwise to protein solution (150 mg BSAr OVA in 12 mL of carbonate buffer, 50 mM, pH 9.6), andhe solution was stirred overnight at 4 ◦C. The reaction mix-ure was dialyzed against five changes of PBS for 5 days andtored lyophilized at −20 ◦C.

.4.3. Preparation of chlorimuron-ethyl (CE) immunogennd coating antigen [19]

An amount of 1.5 g of ethyl 2-(aminosulfonyl) benzoate and.6 g of succinic anhydride was dissolved in 15 mL dioxane fol-owed by addition of 4.6 mL of DBU dropwise in 10 min. Theolution was stirred at 22 ◦C for 2 h and acidified slowly with 2 MCl. Then the solution was removed by rotary evaporation. The

esidue was reconstituted in 25 mL of ethyl acetate and washedith distilled water for three times. After the ethyl acetate extractas dried over anhydrous Na2SO4, the solvent was evaporated.he hapten product was recrystallized in ethanol. The hapten

14.4 mg), NHS (18.2 mg) and DCC (32 mg) were dissolved in

82 J. Zhao et al. / Analytica Chimica Acta 571 (2006) 79–85

0.4 mL of DMF. The activation reaction was carried out for 1 h at22 ◦C and for 18 h 4 ◦C. The mixture was centrifuged and 250 �Lof supernatant was added dropwise to BSA solution (50 mg ofBSA in 5 mL PBS). The solution was stirred for 4 h at 4 ◦C. Thereaction mixture was dialyzed against five changes of PBS for5 days and stored at −20 ◦C after lyophilization. The coatingantigen (CE-OVA) was prepared in the same method.

2.5. Immunization

Two New Zealand white rabbits (2–3 kg) were immunizedwith each immunogen. About 2 mg of conjugate dissolved in1 mL PBS was emulsified with 1 mL Freund’s complete adju-vant, and then injected intradermally at multiple sites (3–5 sites)on the back. The immunization using Freund’s incomplete adju-vant was repeated twice with 20 days interval. After the thirdimmunization, the rabbits received an intramuscular boosterinjection with 1 mg conjugate in 1 mL of PBS and bled 7 daysafter the booster injection.

2.6. Conventional icELISA protocol

In all the procedures, microtiter plates were washed with250 �L PBS or PBST per well four times using the automatedplate washer.

For conventional icELISA, a microtiter plate was first coatedwwohttiuw2PPs2r

2

tiOwoiobjsaa

was stopped by adding 100 �L of 2 M H2SO4. Absorbance wasread at 492 nm in the microplate reader.

2.8. Analysis of glycyrrhetinic acid in licorice roots withconventional icELSIA, simplified icELISA and liquidchromatography (LC)

Four licorice roots samples were collected from ShanxiProvince and the Inner Mongolia Autonomous Region, China.The licorice roots were air dried and ground into powder. Anamount of 0.1 g of licorice powder was placed in a flask fol-lowed by addition of 50-mL of deionized water. The sample wasvortex mixed for 30 min at room temperature. The whole sus-pension was then transferred to a centrifuge tube and centrifugedat 3500 rpm. Part of supernatant was diluted with deionizedwater (1:1000) for ELISA detection. In addition, 7 mL super-natant was lyophilized and dissolved in 0.5 mL solution ofmethanol:NH4Ac:HAc (67:33:1). The solution was analyzed byLC [20] after being filtered through a 0.45 �L filter.

3. Results

3.1. Standard curves for ZR, GA and CE obtained usingconventional icELISA and simplified icELISA

All standards were analyzed in four-well replicates in 1 day.Awbi(

(oaOttwipwvssraptw

3c

dI

ith 200 �L hapten-OVA in coating buffer for 3 h at 37 ◦C. Afterashing four times with PBS, the plate was blocked with 200 �Lf 3% non-fat dry milk in PBS for 30 min at 37 ◦C. After the platead been washed with PBST, 20 �L of various concentrations ofhe standard in PBSTG were pipetted into each well followed byhe addition of 180 �L purified anti-hapten IgG solution dilutedn PBSTG. Plates were then incubated for 0.5 h at 37 ◦C. Thenbound antibody was removed by washing the plates four timesith PBST. After incubation of the plates for 0.5 h at 37 ◦C,00 �L goat anti-rabbit IgG-peroxidase conjugate, diluted inBSTG was added to each well. Plates were then washed withBST, which was followed by the addition of 200 �L sub-trate solution. The reaction was stopped by adding 100 �L ofM H2SO4. Absorbance was read at 492 nm in the microplate

eader.

.7. Simplified icELISA protocol

The washing steps in the simplified icELISA were the same ashose described for the conventional icELISA. In the simplifiedcELISA, a microtiter plate was first coated with 200 �L hapten-VA per well in coating buffer for 3 h at 37 ◦C. After beingashed four times with PBS, plates were blocked with 200 �Lf 3% non-fat dry milk in PBS for 30 min at 37 ◦C. After wash-ng the plates again with PBST, 20 �L of various concentrationsf the standard in PBSTG were pipetted into each well followedy the addition of 90 �L goat anti-rabbit IgG-peroxidase con-ugate diluted in PBSTG and 90 �L purified anti-hapten IgGolution diluted in PBSTG. Plates were then incubated for 0.5 ht 37 ◦C. Following incubation, plates were washed with PBSTnd then 200 �L of substrate solution was added. The reaction

total of six repetitions were performed. Checkerboard assaysere used to select the optimal dilutions of coating antigen, anti-ody, and enzyme-labeled secondary antibody for conventionalcELISA and simplified icELISA based on sensitivity (IC50)Table 1).

The inhibition curves for ZR (Fig. 2a), GA (Fig. 2b) and CEFig. 2c) were obtained separately with optimum combinationsf antiserum, coating antigen and the enzyme-labeled secondaryntibody dilutions for conventional and simplified icELISA. TheD response was given in Table 2. These results showed that

he simplified icELISA had a lower limit of detection (LOD)han conventional icELISA. The IC50 value for ZR, GA and CEas 35, 33 and 52 ng mL−1, respectively, by the conventional

cELISA and 26, 27 and 37 ng mL−1, respectively, by the sim-lified icELISA. The LOD (defined as IC20) for ZR, GA and CEas found to be 3, 3 and 5 ng mL−1, respectively, by the con-entional icELISA and 2, 2 and 3 ng mL−1, respectively, by theimplified icELISA. The detection range of conventional andimplified icELISAs was 3–358 and 2–311 ng mL−1 for ZR,espectively; 3–335 and 2–313 ng mL−1 for GA, respectively;nd 5–495 and 3–450 ng mL−1 for CE, respectively. The sim-lified icELISA has a better sensitivity for ZR, GA and CE, andhe IC50 and IC20 values obtained with the two assay formatsere found to be significantly different (p = 0.05) (Table 3).

.2. Analysis of glycyrrhetinic acid in licorice roots withonventional icELSIA, simplified icELISA and LC

The content of glycyrrhetinic acid in licorice roots wasetected with conventional icELISA and simplified icELISA.n addition, the results were validated by LC.

J. Zhao et al. / Analytica Chimica Acta 571 (2006) 79–85 83

Tabl

e1

OD

data

and

%in

hibi

tion

ofze

atin

ribo

side

,gly

cyrr

hetin

icac

idan

dch

lori

mur

on-e

thyl

inco

nven

tiona

licE

LIS

Aan

dsi

mpl

ified

icE

LIS

A

Con

c.(n

gm

L−1

)Z

eatin

ribo

side

Gly

cyrr

hetin

icac

idC

hlor

imur

on-e

thyl

Con

vent

iona

licE

LIS

ASi

mpl

ified

icE

LIS

AC

onve

ntio

nali

cEL

ISA

Sim

plifi

edic

EL

ISA

Con

vent

iona

licE

LIS

ASi

mpl

ified

icE

LIS

A

OD

a(B

0−B

)/B

0(%

)bO

Da

(B0−

B)/

B0

(%)b

OD

a(B

0−B

)/B

0(%

)bO

Da

(B0−

B)/

B0

(%)b

OD

a(B

0−B

)/B

0(%

)bO

Da

(B0−

B)/

B0

(%)b

2.0

×10

30.

02±

0.01

20.

02±

0.01

10.

02±

0.01

10.

02±

0.01

20.

02±

0.02

20.

03±

0.02

31.

0×

103

0.05

±0.

026

0.10

±0.

026

0.06

±0.

025

0.06

±0.

026

0.12

±0.

0112

0.09

±0.

0310

0.5

×10

30.

10±

0.02

110.

21±

0.03

130.

15±

0.04

120.

13±

0.03

130.

21±

0.01

210.

16±

0.03

180.

1×

103

0.31

±0.

0434

0.51

±0.

0431

0.40

±0.

0631

0.31

±0.

0430

0.41

±0.

0342

0.32

±0.

0235

5.0

×10

10.

44±

0.04

470.

63±

0.05

390.

57±

0.08

440.

42±

0.06

400.

56±

0.04

580.

43±

0.03

471.

0×

101

0.65

±0.

0971

0.98

±0.

0960

0.93

±0.

0972

0.65

±0.

0463

0.77

±0.

0879

0.67

±0.

0473

5.0

×10

0.75

±0.

0981

1.28

±0.

0879

1.07

±0.

0983

0.81

±0.

0579

0.84

±0.

0686

0.74

±0.

0580

1.0

×10

0.87

±0.

0994

1.46

±0.

0989

1.18

±0.

0892

0.90

±0.

0587

0.89

±0.

0692

0.82

±0.

0589

0.5

×10

0.90

±0.

0898

1.55

±0.

0495

1.23

±0.

0996

0.98

±0.

0495

0.93

±0.

0695

0.89

±0.

0496

B0

0.92

±0.

0710

01.

63±

0.06

100

1.29

±0.

0910

01.

03±

0.03

100

0.97

±0.

0410

00.

92±

0.05

100

aO

ptic

alde

nsity

(OD

)da

taar

egi

ven

asth

em

ean

±S.

D.

bB

0an

dB

are

OD

inth

eab

senc

ean

dth

epr

esen

ceof

the

smal

lcom

poun

ds,r

espe

ctiv

ely.

Fig. 2. Standard curves of inhibition by zeatin riboside (A), glycyrrhetinic acid(B), and chlorimuron-ethyl (C) in (©) conventional icELISA and (�) simplifiedicELISA.

The correlation between data obtained with conventionalicELISA and simplified icELISA analyses was 0.9888. Therewas no significant difference among the contents obtained bythree methods for each sample (Table 2). The results suggest thatthe simplified icELISA be useful for high throughput screeningof small molecules.

4. Discussion

Three small molecular compounds, namely ZR, GA, andCE, which are a plant hormone, an ingredient of Chinese tra-ditional medicine, and a herbicide, respectively, were used toverify the new assay and compare the results obtained usingthe simplified icELISA with those obtained using conventionalicELISA. There are two major advantages of the simplified

84 J. Zhao et al. / Analytica Chimica Acta 571 (2006) 79–85

Table 2Content of glycyrrhetinic acid (%) in of licorice roots determined with icELISA and LC (the same letter indicates no difference at p = 0.05)

Samples Conventional icELISAa Simplified icELISAa LCa

Biyearly cultivar collected from Shanxi 0.95 ± 0.04 a 1.09 ± 0.03 a 1.04 ± 0.02 aPerennial wild srain collected from collected from Shanxi 3.20 ± 0.08 a 2.93 ± 0.06 a 3.03 ± 0.06 aBiyearly cultivar collected from Inner Mongolia 1.10 ± 0.05 a 1.22 ± 0.04 a 1.17 ± 0.05 aPerennial wild strain collected from Inner Mongolia 4.46 ± 0.06 a 4.53 ± 0.06 a 4.18 ± 0.06 a

a Data are given as the mean ± S.D.; mean of six determinations.

Table 3Comparison of IC50 and IC20 (ng mL−1) between conventional and simplified icELISAs (the different letters a and b indicate significant difference at p = 0.05)

Assay format Zeatin riboside Glycyrrhetinic acid Chlorimuron-ethyl

IC50 IC20 IC50 IC20 IC50 IC20

Conventional icELISA 35a 3 a 33a 3 a 52a 5 aSimplified icELISA 26b 2 b 27b 2 b 37b 3 b

Table 4Comparison of reagent consumption between conventional icELISA and simplified icELISA

Coating antigen Antibody IgG-HRP

S-icELSIA C-icELISA S-icELSIA C-icELISA S-icELSISA C-icELISA

DR V DR V DR V DR V DR V DR V

Zeatin riboside 1/4000 200 1/16000 200 1/1000 90 1/2000 180 1/200 90 1/4000 200R 4.0 1.0 9.0Glycyrrhetinic acid 1/8000 200 1/16000 200 1/250 90 1/500 180 1/125 90 1/500 200R 2.0 1.0 1.8Chlorimuron- ethyl 1/400 200 1/2000 200 1/200 90 1/8000 180 1/125 90 1/250 200R 5.0 20.0 0.9

S-icELISA: simplified indirect competitive ELISA; C-icELISA: conventional icELISA; DR: dilution ratio; V: volume added into the one well (�L); R: (reagentamount used in S-icELISA/reagent amount used in C-icELISA) as in 20 �L well−1 of standard.

icELISA. First, the actual procedure in the new icELISA forsmall molecules has been simplified by changing the protocol;this has resulted in a time saving of at least 0.5 h and elim-ination of one washing step. Second, the simplified icELISAis more sensitive than the conventional icELISA. The LODand IC50 values obtained using the simplified icELISA proce-dure for three compounds examined in the present study weredecreased compared with results obtained using conventionalicELISA. The IC50 of ZR decreased approximately 1.3-fold andthe IC20 decreased approximately 1.5-fold. Relative values forGA showed 1.2- and 1.4-fold decreases, respectively, whereas,IC50 and IC20 values for CE were decreased 1.4- and 1.5-fold,respectively. Although the degree of decrease was not the samefor the three compounds, the differences in IC50 and IC20 val-ues determined with the two different assays (conventional andsimplified icELISA) were significantly different (Table 3).

However, the amount of antigen, antibody and IgG-HRP usedin the simplified icELISA is much higher than that used in theconventional icELISA (Table 4). The coating antigen is 2–5-fold, the antibody is 1–20-fold, and the IgG-HRP is 0.9–9-fold.Thus, it can be clearly seen that it is more expensive to analyzesamples using the simplified icELISA. The new assay is moreeconomical than competing methods such as GC and LC andis particularly suited to samples that can be analyzed withoutexpensive clean-up.

Many ELISA protocols reported in the literature require thatthe analyte volume added into the plate well to be 50% of thetotal reaction volume [21–24]. However, the sample accountsfor only 10% of the total reaction volume in most commercialELISA kits. In the simplified icELISA described in the presentstudy, the volume of analyte added into the plate well is also10% of the total reaction volume. The main purpose of devel-oping this new procedure was to reduce the effect of the samplematrix.

Acknowledgement

This project was supported by Application and Exten-sion of State Agricultural Technology Achievement Grant No.03EFN217000316.

References

[1] V.S. Morozova, A.I. Levashova, S.A. Eremin, J. Anal. Chem. 60 (2005)202–217 (translated from Zhurnal Analiticheskoi Khimii 60 (2005)230–246).

[2] B.M. Brena, L. Arellano, C. Rufo, M.S. Last, J. Montano, E.E. Cerni,G. Gonzalez-Sapienza, J.A. Last, Environ. Sci. Technol. 39 (2005)3896–3903.

[3] M.A. Dalvie, E. Sinanovic, L. London, E. Cairncross, A. Solomon, H.Adam, Environ. Res. 98 (2005) 143–150.

J. Zhao et al. / Analytica Chimica Acta 571 (2006) 79–85 85

[4] K. Nishi, M. Ishiuchi, K. Morimune, H. Ohkawa, J. Agric. Food. Chem.53 (2005) 5096–5104.

[5] Y. Cao, Y. Lu, S. Long, J. Hong, G. Sheng, Environ. Int. 31 (2005)33–42.

[6] V. Krikunova, G. Hegedus, H.M. Le, L. Jouravleva, S. Eremin, M.Natangelo, E. Benfenati, A. Szekacs, Intern. J. Environ. Anal. Chem.82 (2002) 865–878.

[7] V. Kolar, A. Deng, M. Franek, Food Agric. Immunol. 14 (2002) 91–105.[8] T.D. Sherman, K.C. Vaughn, Weed Sci. 39 (1991) 514–520.[9] A.N. Blintsov, M.A. Gussakovskaya, Biochemistry (Moscow) 69 (2004)

1099–1108 (translated from Biokhimiya 69 (2004) 1353–1364.[10] Z.H. Qiu, J.C. Wu, B. Dong, D.H. Li, H.N. Gu, Crop Prot. 23 (2004)

1131–1136.[11] E.W. Weiler, Ann. Rev. Plant Physiol. 35 (1984) 85–95.[12] S.J. Shan, H. Tanaka, Y. Shoyama, Anal. Chem. 73 (2001) 5784–

5790.[13] M. Mizugaki, K. Itoh, M. Hayasaka, S. Ishiwata, S. Nozaki, N. Nagata,

K. Hanadate, N. Ishida, J. Immunoassay 15 (1994) 21–34.[14] T.R. Glass, H. Saiki, T. Joh, Y. Taemi, N. Ohmura, S.J. Lackie, Biosens.

Bioelectron. 20 (2004) 397–403.

[15] Y.W. Chiu, R.L. Chen, Q.X. Li, A.E. Karu, J. Agric. Food Chem. 48(2000) 2614–2624.

[16] J.M. Van Emon, V. Lopez-Avila, Anal. Chem. 64 (1992) 79–88.[17] F. Jung, S.J. Gee, R.O. Harrison, M.H. Goodrow, A.E. Karu, A.L. Braun,

Q.X. Li, B.D. Hammock, Pestic. Sci. 26 (1989) 303–317.[18] E.M.S. Macdonald, D.E. Akiyoshi, R.O. Morris, J. Chromatogr. 214

(1981) 101–109.[19] J.M.A. Schlaeppi, W. Meyer, K.A. Ramsteine, J. Agric. Food Chem. 40

(1992) 1093–1098.[20] Pharmacopoeia Commission of People’s Republic of China, Pharma-

copoeia of People’s Republic of China, vol. 1, Chemical Industry Press,Beijing, 2000, pp. 65–66.

[21] Z.Q. He, Z.H. Shi, J. Yan, M.P. Zhao, Z.Q. Guo, W.B. Chang, Talanta65 (2005) 621–626.

[22] M.J. Moreno, A. Abad, A. Montoya, J. Agric. Food Chem. 49 (2001)72–78.

[23] N. Lee, C.K. Holtzapple, M.T. Muldoon, S.S. Deshpande, L.H. Stanker,Food Agric. Immunol. 13 (2001) 5–17.

[24] Y. Suzuki, N. Shimada, R. Niwa, M. Yokoi, M. Nakajima, N. Murofushi,I. Yamaguchi, Biosci. Biotechnol. Biochem. 63 (1999) 648–654.