ANALYTICALBIOCHEMISTRY

Analytical Biochemistry 357 (2006) 9–14

www.elsevier.com/locate/yabio

Hapten synthesis for enzyme-linked immunoassay of the insecticide triazophos

Wen Jun Gui, Ren Yao Jin, Ze Li Chen, Jing Li Cheng, Guo Nian Zhu ¤

Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029, China

Received 27 March 2006Available online 7 August 2006

Abstract

Two haptens of the insecticide triazophos (O,O-diethyl O-[1-phenyl-1H-1,2,4-triazol-3-yl] phosphorothioate) were synthesized byintroducing appropriate spacers in the O-ethyl site of the analyte molecular structure. First, thiophosphoryl chloride (PSCl3) reacts withmethanol at low temperature to give O-ethyl dichlorothiophosphate. After reacting with 1-phenyl-3H-1,2,4-triazol, the O-ethyl dichloro-thiophosphate was transformed into the intermediate O-ethyl O-(1-phenyl-1H-1,2,4-triazol-3-yl) chlorothiophosphate. Then the interme-diate reacts with 4-aminobutyric acid and 6-aminobutyric acid to produce hapten I and hapten II, respectively. The molecule structures ofthe two haptens were identiWed by 1H nuclear magnetic resonance spectrum and mass spectrum. An enzyme-linked immunosorbent assay(ELISA) based on monoclonal antibody was also developed to evaluate the two haptens. Results showed that the monoclonal antibodieswith high titers were obtained after immunizing with protein conjugates of these haptens and that the immunoassay has high aYnity andspeciWcity to triazophos. These results suggested that the haptens were synthesized successfully and could be used for immunoassay forthe rapid screening and sensitive determination of this insecticide.© 2006 Elsevier Inc. All rights reserved.

Keywords: Triazophos; Hapten; Synthesis; Monoclonal antibody; ELISA

Triazophos (O,O-diethyl O-(1-phenyl-1H-1,2,4-triazol- The gas chromatography (GC)1 method has been well

3-yl) phosphorothioate) is an organophosphorus insecti-cide applied to control insects, acarids, and some kinds ofnematode. It is widely used on a variety of crops in Chinaand now is one of the most important pesticides for con-trolling bollworm on paddy Weld in the Yangzi River area.Triazophos is moderately toxic to mammalian species but ishighly toxic to Wsh and bees [1]. Due to its widespread usein agriculture, potential health hazards of triazophos resi-due have been recognized by people and local governmentsduring recent years [2–7].

0003-2697/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.07.023

* Corresponding author. Fax: +86 571 86971220.E-mail address: zhugn@zju.edu.cn (G.N. Zhu).

1 Abbreviations used: GC, gas chromatography; ELISA, enzyme-linked imtography; HRP, horseradish peroxidase; IgG, immunoglobulin G; OVA, ovathymidine; HT, hypoxanthine and thymidine; NHS, N-hydroxysuccinimide; Dn-butylamine; TEA, triethylamine; NMR, nuclear magnetic resonance; DMSOEI, electron ion; TZM-1, O-ethyl dichlorothiophosphate; TZM-2, O-ethyl Oane; THBu, O-ethyl O-(1-phenyl-1H-1,2,4-triazol-3-yl) N-(3-carboxypropyl) p1H-1,2,4-triazol-3-yl) N-(5-carboxyamyl) phosphoramidothioate; PBS, phosp

established for the analysis of triazophos in agriculturaland environmental samples [5–7]. But it is time-consum-ing and requires sophisticated equipment in a laboratory.Immunoassays are demonstrated as a simple, rapid, andcost-eVective alternative to those traditional methods incases where high-throughput and/or on-site screeninganalysis are needed [8,9]. Here we report the synthesis ofhaptens for triazophos and the development of anenzyme-linked immunosorbent assay (ELISA) for the

munosorbent assay; McAb, monoclonal antibody; TLC, thin-layer chroma-lbumin; BSA, bovine serum albumin; HAT, hypoxanthine, aminopterin, and

CC, N,N�-dicyclohexylcarbodiimide; OPD, O-phenylenediamine; TBA, tri-, dimethyl sulfoxide; TMS, tertramethylsilane; ESI, electrospray ionization;

-(1-phenyl-1H-1,2,4-triazol-3-yl) chlorothiophosphate; DCM, dichlorometh-hosphoramidothioate; RT, room temperature; THHe, O-ethyl O-(1-phenyl-

hate-buVered saline; CR, cross-reactivity; LOD, limit of detection.

10 Hapten synthesis for ELISA of triazophos / W.J. Gui et al. / Anal. Biochem. 357 (2006) 9–14

analyte based on monoclonal antibodies (McAbs) toevaluate the haptens.

Materials and methods

Reagents and instruments

All reagents were of analytical grade unless speciWedotherwise. Triazophos and other pesticide standards wereobtained from National Standards (China). Starting prod-ucts for hapten synthesis were kindly provided by Hangz-hou Pesticide (China). Thin-layer chromatography (TLC)was carried out on 0.2 mm precoated silica gel F254 (100–200 mesh, particle size) on glass sheets. Enzyme immunoas-say-grade horseradish peroxidase (HRP); goat anti-mouseimmunoglobulin G (IgG) HRP; ovalbumin (OVA, MW45,000); bovine serum albumin (BSA, MW 67,000); culturemedia RPMI-1640, hypoxanthine, aminopterin, andthymidine (HAT), and hypoxanthine and thymidine (HT);N-hydroxysuccinimide (NHS); and N,N�-dicyclohexylcar-bodiimide (DCC) all were obtained from Sigma (Spain).Complete and incomplete Freund’s adjuvants wereobtained from the Institute of Bioproducts (China). TheSP2/0 mouse plasmacytoma line was obtained from theShanghai Institute of Cell Biology (China). O-Phenylenedi-amine (OPD), tri-n-butylamine (TBA), triethylamine(TEA), isobutyl chloroformate, and other chemicalreagents were obtained from Shanghai Chemical Reagents(China). The ELISA was carried out in 96-well polystyrenemicroplates (Costar, USA).

1H nuclear magnetic resonance (NMR) spectra wereobtained with an Avance DMX 500 spectrometer (Bruker,Germany) operating at 400 MHz for solutions in dimethylsulfoxide (DMSO)–D6. Chemical shifts are given relative totertramethylsilane (TMS). Electrospray ionization (ESI)mass spectra were obtained on an Esquire mass spectrome-ter (Bruker, Germany). Electron ion (EI) mass spectra wereobtained on an HP 5890/5973 mass spectrometer (Hewlett–Packard, USA). UV–vis spectra were recorded on a spec-trophotometer (Xinmao, China). ELISA plates werewashed with a DEM plate washer (Beijing Tuopu Analyti-cal Instruments, China). Well absorbencies were read with amodel 550 plate reader (Bio-Rad, USA).

Hapten synthesis

Two haptens were prepared by introducing amino acidspacers with diVerent C-chain lengths in the O-ethyl site oftriazophos molecular structure (Fig. 1).

Synthesis of O-ethyl dichlorothiophosphateO-Ethyl dichlorothiophosphate (TZM-1) was synthe-

sized according to Ref. [10]. BrieXy, to a strongly stirredliquid of thiophosphoryl chloride (34.0 g, 0.4 mol) cooledin an ice bath was added methanol (35 ml, 0.6 mol) drop-wise over 1 h. After 30 min of stirring at less than 5 °C, themixture was washed with 75 ml (3£ 25 ml) water (pre-cooled to less than 5 °C), and then the water wasremoved. The residue was dried over anhydrous MgSO4overnight and Wltered, and then the Wltrate was distilledin decompression. The distillation of boiling point 60–61 °C/0.106 kPa was collected to give TZM-1 (25.9 g,73.2%) as colorless oil. Product structure was conWrmedby spectral data: EI-MS, m/z (relative intensity) 180 (43,M+ + 2), 178 (70, M+), 152 (62), 150 (91), 117 (33), 115(100).

Synthesis of O-ethyl O-(1-phenyl-1H-1,2,4-triazol-3-yl) chlorothiophosphate

Intermediate O-ethyl O-(1-phenyl-1H-1,2,4-triazol-3-yl)chlorothiophosphate (TZM-2) was prepared accordingto Refs. [11,12]. To a stirred solution of 1-phenyl-3H-1,2,4-triazol (16 g, 0.1 mol) dissolved in 80 ml dichloro-methane (DCM) and 15 ml TEA cooled in an ice bathwas added O-ethyl dichlorothiophosphate (36.0 g,0.2 mol). Then 2.3 mol/L solution of NaOH (50 ml) wasadded to the mixture dropwise over 30 min. After 1 h ofstirring at less than 10 °C, the mixture was washed with5% solution of NaOH (3£ 25 ml) and then with distilledwater (3£ 50 ml). The organic phase was dried over anhy-drous MgSO4 and Wltered, and the Wltrate was concen-trated under reduced pressure. The residue was extractedwith petroleum ether (3£ 50 ml), and the extract wasconcentrated under reduced pressure to give TZM-2(10.6 g, 35%) as fawn oil: TLC Rf 0.81 (EtAc/hexane, 1:2);ESI-MS, m/z (relative intensity) 304 (100, [M + 1]+), 306(33, [M + 2 + 1]+).

Fig. 1. Reaction schemes of synthesis for haptens of triazophos.

P

S

Cl

P Cl

S

Cl

ClC2H5OH

-5 ¡æ

N N

NHO

NaOH

P

SCl

C2H5O

N N

NO

Cl

OC2H5

NH2CH2(CH2)nCOOH

(n=2, 4)

P

SC2H5O

HN

N N

NO

CH2(CH2)nCOOH

(TZM-1)

(TZM-2)

(Haptens)

Hapten synthesis for ELISA of triazophos / W.J. Gui et al. / Anal. Biochem. 357 (2006) 9–14 11

Synthesis of O-ethyl O-(1-phenyl-1H-1,2,4-triazol-3-yl) N-(3-carboxypropyl) phosphoramidothioate

In preparing O-ethyl O-(1-phenyl-1H-1,2,4-triazol-3-yl) N-(3-carboxypropyl) phosphoramidothioate (THBu),to 10 ml stirred aqueous solution containing 4-aminobu-tyric acid (1.03 g, 10 mmol) and NaOH (0.4 g, 10 mmol)cooled in an ice bath was added TZM-2 (1.51 g, 5 mmol,diluted in 5 ml of 4,4-dimethyl-1,3-dioxane) dropwise.Subsequently, 10 ml aqueous solution of NaOH (1 mol/L)was added dropwise, and then the ice bath was takenaway. After stirring at room temperature (RT) for 1 h, themixture was washed with petroleum ether/acetate ester(9:1, 3£ 10 ml). The water phase was adjusted to pH 3with 2 mol/L HCl and then was extracted with ethyl ace-tate (3£ 20 ml). The extract was dried over anhydrousMgSO4, and the solvent was evaporated to give the rudeproduct as a white solid. This solid was recrystalled inEtAc/hexane (1:9) to give THBu as clustered colorlesscrystal (0.51 g, 27%). TLC Rf 0.65 (EtAc/hexane/formylacid, 50:50:1); ESI-MS, m/z (relative intensity) 371 (100,[M + 1]+); 1H NMR(400 MHz, CDCl3): � 1.33 (t, 3 H,JD 6.8, –CH2CH3), 1.75 (m, 2H, –CH2CH2CH2), 2.33(m, 2H, –CH2COOH), 3.07 (m, 2H, –NHCH2), 4.16(m, 2H, –OCH2CH3), 7.43 (t, 1H, –ArH), 7.57 (t, 2H, –ArH),7.81 (d, 2H, JD8.4, –ArH), 9.08 (s, 1H, NBCH), 11.88 (s, 1H,–COOH).

Synthesis of O-ethyl O-(1-phenyl-1H-1, 2, 4-triazol-3-yl) N-(5-carboxyamyl) phosphoramidothioate

The synthesis of O-ethyl O-(1-phenyl-1H-1,2,4-triazol-3-yl) N-(5-carboxyamyl) phosphoramidothioate (THHe) wasthe same as the synthesis of THBu but starting from 6-aminohexylic acid. TLC Rf 0.73 (EtAc/hexane/formyl acid,50:50:1); ESI-MS, m/z (relative intensity) 399 (100,[M + 1]+); 1H NMR (400 MHz, CDCl3): � 1.28 (t, 3H,–CH2CH3), 1.33 (m, 2H, CH2CH2CH2CH2CH2), 1.49 (m,2H, –CH2CH2COOH), 1.53 (m, 2H, –NH CH2CH2), 2.19(t, 2H, CH2COOH), 3.03 (m, 2H, –NHCH2), 4.22 (m, 2H,–OCH2CH3), 7.43 (t, 1H, –ArH), 7.57 (t, 2H, –ArH), 7.80(t, 2H, –ArH), 9.08 (s, 1H, NBCH), 11.83 (s, 1H, –COOH).

Preparation of hapten–protein conjugate

Immunogens (hapten–BSA conjugates) were preparedby the NHS method, and coating antigens (hapten–OVAconjugates) and enzyme tracer (hapten–HRP) were pre-pared by the isobutyl chloroformate method [13]. Conju-gate formations were conWrmed spectrophotometrically.Molar ratios of approximately 14.6, 8.2, 12.8, 9.4, 2.1, and2.3 were estimated for THBu–BSA, THHe–BSA, THBu–OVA, THHe–OVA, THBu–HRP, and THHe–HRP conju-gates, respectively.

Preparation of McAbs

The McAbs for triazophos were prepared analogouslyto the method of Garrett and coworkers [14]. BrieXy,

BALB/c female mice (8–10 weeks of age) were immunizedsubcutaneously with BSA–THBu or BSA–THHe conju-gate (100 �g) in phosphate-buVered saline (PBS) and com-plete Fround’s adjuvant 1:1 (v/v) initially andsubsequently using incomplete Fround’s adjuvant at 2week intervals three times. One week after the last injec-tion, mice were tail bled to check for antibody activity.Once this was found to be positive, the mouse was givenone more booster injection with 100 �g of conjugate inPBS (without adjuvant), it was killed 3 days later, itsspleen was removed, and the spleen cells were fused withSP2/0 murine myeloma cells. Cell fusion procedures werecarried out essentially as described by Nowinski andcoworkers [15]. Culture supernatants were screened forthe presence of antibodies that recognized triazophos 12–14 days after cell fusion. The screening consisted of thesimultaneous performance of noncompetitive and com-petitive indirect ELISAs to test the ability of antibodies tobind the hapten–OVA conjugate and to recognize triazo-phos. Selected hybridomas were cloned by limiting dilu-tion using HT media on a feeder layer of BALB/cthymocytes (»106 cells/well) and peritoneal macrophages(»5000 cells/well). Stable antibody-producing clones wereexpanded and cryopreserved in liquid nitrogen. After titertesting with indirect ELISA of the mice ascites, theMcAbs were separated by the method of salting out (withcaprylic acid–ammonium sulfate) as described by Zhuand coworkers [13] and then were puriWed by an Immuno-Pure (A) IgG puriWcation kit (Pierce, USA).

Screening ELISA

For competition assays, the concentrations of antibodiesand enzyme tracer were optimized by checkerboard titra-tion. ELISA plates were coated with antibody solutions (inPBS). Standards were prepared in PBS1 (PBS containing274 mM NaCl) and methanol (PBS1:methanol, 9:1, v/v) byserial dilutions from a stock solution in methanol usingborosilicate glass tubes. All incubations were carried out at37 °C. After each incubation, plates were washed four timeswith PBST (PBS containing 0.05% Tween 20, pH 7.4).Competitive curves were obtained by plotting absorbanceagainst the logarithm of analyte concentration. Sigmoidcurves were simulated by means of Microsoft Excel 2000.

Microtiter plates were coated with the optimized con-centrations of antibodies by incubation for 2 h. The plateswere washed and then blocked by incubation with 2% deg-reasant milk in PBS (300�l/well) for 30 min. After anotherwash step, 100 �l/well of hapten–HRP conjugate previouslydiluted with PBS1 was added. After incubation for 1 h, theplates were washed and 100�l/ml of OPD solution (10 mgof OPD and 10�l of 30% H2O2 diluted with 25 ml of phos-phate–citrate buVer, pH 5.4) was added. After incubationfor 15 min, the reaction was stopped by adding 50 �l of 2 MH2SO4, and absorbance at 490 nm was read and recorded.For competitive assays, the procedure was the same exceptthat after blocking, a competition step was introduced by

12 Hapten synthesis for ELISA of triazophos / W.J. Gui et al. / Anal. Biochem. 357 (2006) 9–14

adding 50�l per well of the competitor followed by 50 �lper well of the proper concentration of HRP-labeled hap-ten in 4% degreasant milk–PBS1.

Table 1Titer of ascites using homologous and heterologous haptens for coatingconjugates

a Ascites dilution was considered suitable while OD490 nm value wasapproximately 1.0. Boldface numbers indicate heterologous assays.

Immunizing hapten Titera

Coated with THBu–OVA

Coated with THHe–OVA

THBu 1.28 £ 106 2.56£ 106

THHe 1.02 £ 107 5.12 £ 106

Table 2AYnity to triazophos using direct competitive ELISA format

For antibody-coating format, optimal conditions were selected.a Triazophos concentration reducing the ELISA maximum response to

50%. Data represent the means of six experiments. Boldface numbers indi-cate heterologous assays.

Coating antibody I50 (ng/ml)a

THBu–HRP THHe–HRP

THBu McAb 1.84 1.08THHe McAb 0.65 1.76

Results and discussion

Hapten selection and synthesis

A suitable hapten and its artiWcial antigen for immuniza-tion should preserve the structure of the target compoundas much as possible. Commonly, to obtain a high selectivityand sensitivity of an antibody against a given phosphoro-thioate organophosphorus (OP) pesticide, it was suggestedto synthesize hapten having a spacer arm at the thiophos-phate group but preserving the group of aromatic rings orheterocycles because for most organophosphorus pesticidesthe thiophosphate group has a common structure thatdiVers only in the structure of the aromatic rings or hetero-cycles [16]. To synthesize this kind of hapten, a generalmethod was developed by McAdam and Skerritt [17]. Thismethod involved seven reaction steps. Several haptens wereprepared based on this method [17–22]. A simpliWed syn-thetic route for chlorpyrifos hapten was developed by Choand coworkers [11]. Recently, the immunoassay for triazo-phos based on polyclonal antibody was described by Liuand coworkers [23], but they did not report the synthesismethod for the haptens of triazophos. Therefore, two hap-tens (THBu and THHe) for triazophos ELISAs in thisstudy were synthesized analogously to the method of Cho

Table 3SpeciWcity of triazophos McAb derivatizing from hapten THHe using antibody-coated ELISA format

a Percentage of cross-reactivity D (I50 of triazophos/I50 of other compound) £ 100. Standards were diluted in PBS1 and methanol (PBS1:methanol,9:1, v/v).

Compound Structure CR (%)a

THBu–HRP THHe–HRP

Triazophos 100 100

Chlorpyrifos <0.01 <0.01

Diazinon 0.03 0.07

Malathion <0.01 <0.01

Parathion <0.01 <0.01

Fenitrothion <0.01 <0.01

N N

NOP

SC2H5O

C2H5O

N Cl

ClCl

OP

S

C2H5OC2H5O

N

NOP

SC2H5O

C2H5O

CH3

CH

CH3

CH3

PCH3O

CH3O

S

S CH

C

O

O CH2CH3

CH2

C

O

O CH2CH3

NO2OPC2H5O

C2H5O

S

SCH3

CH3

OP

SMeO

MeO

Hapten synthesis for ELISA of triazophos / W.J. Gui et al. / Anal. Biochem. 357 (2006) 9–14 13

and coworkers [11]. From the data of MS and 1H NMR, weknow that the products were the goals we expected.

Immune response to conjugates of triazophos hapten

Homologous and heterologous ELISAs in indirect for-mat were used to characterize for reactivity of the McAbsto hapten conjugates (Table 1). Results showed that miceascites displayed a high level for each relative homologousand heterologous hapten conjugates with favorable titers(1.28£106–1.02£107).

Characterization of McAbs

AYnity to triazophos of McAbs was characterized usinghomologous and/or heterologous hapten in the antibody-coated ELISA format (Table 2). Results showed that theMcAb from hapten THHe (THHe McAb)/THBu–HRPcombination displayed higher aYnity with an I50 value of0.65 ng/ml. Results also showed that for THHe McAb, thesensitivity of the heterologous assays was nearly threefoldbetter than that of the homologous assays. The THHeMcAb was selected for speciWcity investigation by anti-body-coated direct ELISA format due to its high aYnity totriazophos. Several other organophosphorus insecticides oranalogues were tested for cross-reactivities (CRs) to evalu-ate the speciWcity of the THHe McAb. The detailed CRdata are summarized in Table 3. Results showed that theinterference was negligible under the optimized conditionof ELISA.

Competitive curve of triazophos standards

A typical standard curve of the triazophos immunoassayis given in Fig. 2. The average I50 for triazophos was0.65 ng/ml. The limit of detection (LOD) was nearly 0.10 ng/ml, which established as the analyte concentration showing80% of the maximum response (20% inhibition). The work-ing range was assigned to a concentration giving 15–95%

Fig. 2. Standard curve. Conditions: 8000 ng/ml THHe McAb and 200 ng/ml THBu–HRP; standards diluted in PBS containing 274 mM NaCl and10% methanol. Data represent the means of Wve determinations.

inhibition (i.e., 0.06–15.63 ng/ml triazophos), according tothe linear range of the curve. Compared with the anti-tria-zophos polyclonal antibody as described by Liu andcoworkers [23], the THHe McAb has the greater aYnityand sensitivity (the polyclonal antibody displayed aYnitywith an I50 value of 5.51 ng/ml, and the LOD value was0.11 ng/ml, with the linear range being 1–1000 ng/ml).

In summary, the haptens were synthesized successfullyand could be used for immunoassay for the rapid screeningand sensitive determination of this insecticide.

Acknowledgment

This study was supported by the National NaturalScience Foundation of China (30370944).

References

[1] Q.K. Liu, G.N. Zhu, New Manual of Pesticides, Shanghai Science andTechnology Press, Shanghai, China, 1999.

[2] X.S. Li, H.H. Tan, H.Y. Huang, M.Z. Lin, Z.X. Lu, Studies on triazophosresidue in lichee and soil, S.W. China J. Agric. Sci. 17 (2004) 334–336.

[3] F. Yu, D.J. Cai, Z.J. Shan, L.L. Shi, H.G. Zuo, Residue trials on 20%triazophos EC in rice Weld, J. Agro-Environ. Sci. 23 (2004) 384–386.

[4] Y. Wang, G.L. Wang, H. Yu, Remainder and change trender of tria-zophos in the body of bred Legillarca granosa Linnaeus, Dong HaiMar. Sci. 22 (2004) 35–39.

[5] D.X. Gong, L.Y. Zheng, R.B. Yang, Z.Y. Guo, Z.M. Zhou, Analyticalmethod of residue of triazophos insecticide in citrus: water and soilwith gas chromatography, J. Agro-Environ. Sci. 23 (2004) 1034–1036.

[6] Y.L. Ji, G.Q. Lin, D.B. Zhou, Z.Y. Huang, L. Li, L. Liu, Residualdetermination of triazophos in vegetable by gas chromatography,Pesticides 36 (1997) 30–31.

[7] S.B. Zhang, J. Yi, J.L. Ye, W.H. Zheng, X.Q. Cai, Z.B. Gong, Determi-nation of buprofezin, methamidophos, acephate, and triazophos resi-dues in Chinese tea samples by gas chromatography, ChinaJ. Chromatogr. 22 (2004) 154–157.

[8] J.P. Sherry, Environmental chemistry: the immunoassay option, Crit.Rev. Anal. Chem. 23 (1992) 217–300.

[9] E.P. Meulenberg, W.H. Mulder, P.G. Stoks, Immunoassays for pesti-cides, Environ. Sci. Technol. 29 (1995) 553–561.

[10] P.Y. Peng, C.Y. Jiang, Y.P. Wang, Synthesis of O-ethyl dichlorothio-phosphate and the reaction kinetics, Chin. J. Appl. Chem. 18 (2001)257–258.

[11] Y.A. Cho, H.S. Lee, E.Y. Park, Y.T. Lee, B.D. Hammock, K.C. Ahn,J.K. Lee, Development of an ELISA for the organophosphorus insec-ticide chlorpyrifos, Bull. Korea Chem. Soc. 23 (2002) 481–487.

[12] Z.G. Li, Preparation of Organointermediates, second ed., ChemicalIndustry Press, Beijing, China, 2001.

[13] G.N. Zhu, G. Wu, H.M. Wu, Synthesis and identiWcation of the anti-gens for the organophosphorus insecticide chlorpyrifos, Sci. Agric.Sin. 36 (2003) 657–662.

[14] S.D. Garrett, D.J.A. Appleford, G.M. Wyatt, H.A. Lee, M.R.A. Mor-gan, Production of a recombinant anti-parathion antibody (scFv):stability in methanolic food extracts and comparison to an anti-parathion monoclonal antibody, J. Agric. Food Chem. 45 (1997)4183–4189.

[15] R.C. Nowinski, M.E. Lostrom, M.R. Tam, M.R. Stone, W.N. Bur-nette, The isolation of hybrid cell lines producing monoclonal anti-bodies against the P15(e) protein of ecotropic murine leukemiaviruses, Virology 93 (1979) 111–126.

[16] K.O. Kim, Y.J. Kim, Y.T. Lee, B.D. Hammock, H.S. Lee, Develop-ment of an enzyme-linked immunosorbent assay for the organophos-phorus insecticide bromophos-ethyl, J. Agric. Food Chem. 50 (2002)6675–6682.

14 Hapten synthesis for ELISA of triazophos / W.J. Gui et al. / Anal. Biochem. 357 (2006) 9–14

[17] D.P. McAdam, J.H. Skerritt, Synthesis of organothiophosphate anti-gens for the development of speciWc immunoassays, Aust. J. Chem. 46(1993) 959–967.

[18] S.L. Edward, J.H. Skerritt, A.S. Hill, D.P. McAdam, An improvedimmunoassay for chlorpyrifos-methyl (Reldan) in grain, Food Agric.Immunol. 5 (1993) 129–144.

[19] A.S. Hill, J.H. Skerritt, R.J. Bushway, L.B. Perkin, K. Larkin, M.Thomas, W. Korth, K.H. Bowmer, Development and application oflaboratory and Weld immunoassays for chlorpyrifos in water and soilmatrices, J. Agric. Food Chem. 42 (1994) 2051–2058.

[20] J.J. Manclus, J. Primo, A. Montoya, Development of enzyme-linkedimmunosorbent assays for the insecticide chlorpyrifos: I. Monoclonal

antibody production and immunoassay design, J. Agric. Food Chem.44 (1996) 4052–4062.

[21] D.P. McAdam, A.S. Hill, H.L. Beasley, J.H. Skerritt, Mono- andpolyclonal antibodies to the organophosphate fenitrothion:I. Approaches to hapten–protein conjugation, J. Agric. Food Chem.40 (1992) 1466–1470.

[22] J.H. Skerritt, A.S. Hill, H.L. Thwaites, S.L. Edward, D.P. McAdam,Enzyme-immunoassay for quantiWcation of the organophosphate pesti-cides: fenitrothion, chlorpyrifos-methyl, and pyrimiphos-methyl in wheatgrain and Xour milling fractions, J. AOAC Intl. 75 (1992) 519–528.

[23] S.Z. Liu, L. Wang, L.H. Wei, Studies on the immunoassay for triazo-phos, China J. Anal. Chem. 33 (2005) 1697–1700.