C o m m u n i c a t i o n s

Enzyme immunoassay for the determination of theinsecticide fenoxycarb

Gianfranco Giraudi*a, Cristina Giovannolia, Claudio Baggiania, Ilaria Rossoa, Paola Colettoa,Marcello Dolcib Gianpaolo Grassic and Adriano Vanniaa Dipartimento di Chimica Analitica, Universita di Torino, Via Giuria 5, 10125 Torino, Italy b Dipartimento di Valorizzazione e Protezione delle Risorse Agroforestali, Universita di Torino,Torino, Italyc Istituto Sperimentale per le Colture Industriali, Bologna, Italy

A very selective polyclonal antiserum against ethyl2-(4-phenoxyphenoxy)ethylcarbamate (fenoxycarb) wasraised in a rabbit by immunization with a2-(4-phenoxyphenoxy)ethanol hemisuccinate–bovineserum albumin conjugate. The antiserum was used tocarry out a heterogeneous competitive enzymeimmunoassay for the selective quantification offenoxycarb at the ng ml21 level. The effects on the assayperformance of different pH and ionic strength values, aswell as the presence of methanol as organic modifier inthe diluent buffer formulation, were studied. Theoptimized calibration curve showed a sensitivity of 0.42ng ml21. The assay selectivity was found to be good, withno significant cross-reactivity towards fenoxycarb-relatedpesticides such as chloroxuron, cypermethrin, diclofop,p,pA-DDT, difenoxuron, permethrin and pyriproxyfen.

Fenoxycarb, ethyl 2-(4-phenoxyphenoxy)ethylcarbamate, is aselective insecticide which acts as an insect growth regulatorand disrupts the development of the pest. It has an insect-specific mode of action exhibiting strong juvenile hormoneactivity. Owing to its effectiveness, to its presumed rapiddegradation in water, soil and plants, and to its low mobility inthe ground, fenoxycarb is widely used in fruit-growing againstvarious fruit-infesting insects.1,2 Nevertheless, the beginning(1988) of the use of fenoxycarb as a fruit plant protector allowedby the Italian Agriculture Department, coincided with theinability of Bombyx mori larvae to spin their cocoon and pupate,as previously observed in other European countries,3 causing aconsiderable economical damage to silk-worm breeding. Thisanomalous behaviour has recently led to careful monitoring,both of the spreading and of the determination of theenvironmental contamination levels in the areas and during theperiods involved in treatments. The results obtained will definethe future use of the pesticide.4 The recommended method forthe determination of fenoxycarb residues is based on solventextraction of the samples with acetone, dilution with water ofthe concentrated extracts, back-extraction with hexane, clean-up on florisil, silica gel or Sep-Pak C18, evaporation of theeluates and analysis of the residues by reversed-phase high-performance liquid chromatography (mobile phase: phosphatebuffer–acetonitrile 1 + 1 v/v).2 In order to make the work-upprocedures less extensive and time-consuming and to attempt asignificant improvement in the analytical sensitivity, weconsidered the immunoassay technique, based on the com-petitive binding to a specific antiserum. This method hasrecently been noted for often being a valid alternative to thetraditional instrumental techniques in residue analysis, above allwhen there is the need to carry out an extensive monitoringinvolving analytical determinations on wide numbers ofsamples.5

The following report deals with the development of apreliminary immunoassay procedure for the direct determina-tion of fenoxycarb residues on aqueous samples.

Experimental

Reagents

Chloroxuron, cypermethrin, diclofop, p,pA-DDT, difenoxuron,fenoxycarb, permethrin and pyriproxyfen were obtained fromDr. Ehrenstorfen GmbH (Augsburg, Germany). Phenol, p-cresol, ethoxybenzene, bovine serum albumin (BSA) andbovine tyreoglobulin (bTG) were obtained from Sigma (St.Louis, MO, USA). The goat anti-rabbit IgG–horseradishperoxidase conjugate was obtained from BioRad (Hercules,CA, USA). All other chemicals were obtained from Merck(Darmstadt, Germany).

2-(4-Phenoxyphenoxy)ethanol hemisuccinate (Fx-HS) waschosen as a fenoxycarb-derived structure able to elicit a properimmunoresponse in immunized rabbits and to constitute asuitable solid phase to perform the immunoassay. This moleculewas synthesised starting from 4-phenoxyphenol (obtained fromSigma) by reaction with ethylene chlorohydrine,6 and sub-sequent hemisuccination using a general method carried out inthe literature.7

Conjugates between proteins and Fx-HS were prepared usingthe N-hydroxysuccinimide-activated ester method.8 Theimmunogen was obtained from the reaction between BSA andFx-HS with a molar reaction ratio of 1 : 300. Coating antigenwas prepared reacting bTG with Fx-HS with a molar reactionratio of 1 : 20. The raw conjugates were purified by low pressuregel filtration on Sephadex G25. The protein fraction wascollected and stored at 230 °C. Protein concentration wasdetermined by use of the Bradford Method.9

Antiserum

The immunization procedure was carried out on one rabbit byfive boosting doses administered at about three week intervalsfor 2 months, except the last dose which was injected after a 3month resting interval. The same amount of antigen (0.2 mg)was used except for the last dose, where the used antigenamount was 0.3 mg. Each immunization step was carried out bymultiple subcutaneous injections in different positions, whilethe fourth boost dose was administered directly into the vein.The subcutaneous injected solutions were prepared by emulsi-fying the Fx-HS–BSA conjugate with an adjuvant in a salinesolution. The adjuvant was the Freund’s complete adjuvant forthe first boosting dose, the Hunter’s adjuvant for the second andthird doses and the Freund’s incomplete adjuvant for the lastdose. Test bleedings for titer determination were conducted twoweeks after each booster.

The antiserum used in the assay procedures (last bleeding)was purified to remove the anti-BSA antibodies by low-pressure

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affinity cromatography on Affiprep 10-BSA according to thedescribed procedure.10 Aliquots of 0.2 ml of purified antiserumwere collected, mixed and stored at –30 °C.

Competition curves

A mixture of 0.1 ml of competitor (fenoxycarb or its analogues,at the concentrations of 0, 0.1, 0.2, 0.4, 2, 4, 20, 40, 200, 400 and800 ng ml21) in phosphae–citrate buffer solution (citric acid–sodium phosphate 0.1 mol l21, sodium chloride in the range of0.05–0.4 mol l21, EDTA 1 mmol l21, gelatine 0.1% m/v, pH inthe range 5.5–7.4) and 0.1 ml of the antiserum (diluted1 : 100 000) was dispensed in duplicate into each well, coatedwith 0.3 ml of Fx-HS–bTG, according to the literature.11 Then,wells were incubated overnight at room temperature. Non-specific binding and the absorbance at the zero concentration ofthe competitor were measured by replacing, respectively, theantiserum and the competitor with the corresponding diluentbuffer. Wells were washed three times with the washingsolution, then the antibodies to fenoxycarb on the solid phasewere revealed by adding 0.2 ml of goat anti-rabbit IgG–horseradish peroxidase conjugate (diluted 1 : 4000 with phos-phate buffer 0.02 mol l21, sodium chloride 0.12 mol l21, EDTA1 mmol l21, gelatine 0.1% m/v, pH 7.4), incubating for 1 hourat 37 °C and washing three times. Finally, 0.2 ml of 1 + 1chromogen–substrate mixture (0.02% m/v tetramethylbenzi-dine dihydrochloride–0.005% v/v hydrogen peroxide, citratebuffer, 0.075 mol l21, pH 5) was dispensed into each well andincubated in the dark for 30 min at 37 °C. The colourdevelopment was blocked by the addition of 0.1 ml of 1 mol l21

sulfuric acid, and the absorbance was read at 450 and 415nm.

The competition curves were fitted by using the fourparameter logistic equation, according to the literature.11 Thedetection limit was evaluated as the concentration of fenoxycarbthat gives a significant absorbance equal to the absorbance ofthe zero standard (A0) minus 3 standard deviations, calculated asthe mean value of twelve repeated experimental measurements.The I50 and I90 were defined as the fenoxycarb concentrationsthat reduce the analytical signal (absorbance) to 50% and to90%, respectively, compared with the curve plateau. Cross-reactivity values were calculated according to the literature.12

Results and discussion

pH Effect

The different pH values examined, as well as the I50, I90 and A0,referred to the corresponding competition curves, are reportedin Table 1. The pH values studied are included in a rangebetween 5.5 and 7.4. The experimental values obtained showedthat a pH decrease of the buffered solution brought aprogressive decrease in the I50 and I90 values, indicating in bothcases a gain in assay sensitivity. Nevertheless, the pH decreasealso causes a clean reduction in the maximum analytical signalon the solid phase, an effect that can compromise the precisionof the fenoxycarb determination because of the smallerabsorbance range in which the calibration curve can beobtained.

Such results induced us to choose a buffer pH value of 7.4 forthe assay development, because this compromise should assurea good sensitivity and a more reliable quantification.

Ionic strength effect

The different ionic strength values examined, as well as the I50,I90 and A0, referred to the corresponding competition curves, arereported in Table 2. As shown by the experimental data theincrease of ionic strength obtained by adding 0.2 mol l21

sodium chloride is the one that assures a more sensitivecompetition curve, while the A0 values do not seem to beinfluenced in a significant way by this parameter. Such a highersensitivity can be due to an increase of the interaction betweenfenoxycarb and antibody binding sites, because of the enhance-ment of hydrophobic interactions.

Methanol effect

Methanol was examined as an organic modifier of the bufferformulation because of its regular use in the extraction of poorlyhydrophilic analytes from samples of environmental origin. Themethanol concentration values examined, as well as the I50, I90and A0, referred to the corresponding competition curves, arereported in Table 3. The experimental data show that limitedamounts of solvent have no significant influence upon thesensitivity of competiton curves, while only with higheramounts (30% v/v) is it possible to observe a definite increaseof the values of I50, I90, with a detrimental effect on the assayperformance.

Antiserum cross-reactivity

The specificity of the assay was tested, taking into account threekinds of potential interferents: the analyte itself and themolecules utilized to prepare both the immunogen and solidphase [fenoxycarb, 2-(4-fenoxyfenoxy)ethanol and fenoxycarbhemisuccinate], some commercial pesticides with structuresrelated to fenoxycarb (reported in Fig. 1), and some otherfenoxycarb-related chemicals (phenol, p-cresol and ethoxy-benzene). The cross-reactivity determined experimentallyshows a high degree of specificity for the antiserum used,because values are all less than 0.1%, with the exception of thepesticide pyriproxyfen (6.0%). The homologous hapten fenoxy-carb hemisuccinate binds antibodies to Fx-HS–BSA better thanfenoxycarb itself by about one order of magnitude (2100%), asa consequence of the heterogeneity of the polyclonal antiserum,in which antibodies able to bind not only the fenoxycarb but alsoother structures, such as the linking bridge used to prepare theimmunogen, are present. This spurious recognition is present,

Table 1 Effect of buffer pH on the fenoxycarb competition curve. Sodiumchloride in diluent buffer: 0.05 mol l21. No methanol present

Buffer pH I50/ng ml21 I90/ng ml21 A0(450 nm)

7.4 9.7 0.39 2.506.5 6.6 0.14 2.006.0 5.3 0.15 1.695.5 2.7 0.054 1.725.0 1.7 0.023 1.17

Table 2 Effect of buffer ionic strength given by various sodium chlorideconcentrations added to phosphate–citrate buffer on the fenoxycarbcompetition curve. Diluent buffer pH 7.4. No methanol present

NaCl/mol l21 I50/ng ml21 I90/ng ml21 A0(450 nm)

0.05 15.2 0.70 2.460.20 9.40 0.35 2.760.40 11.2 0.48 2.39

Table 3 Effect of different methanol additions on the fenoxycarbcompetition curve. Diluent buffer pH 7.4; sodium chloride 0.2 mol l21

MeOH % (v/v) I50/ng ml21 I90/ng ml21 Imax( 415 nm)

0 9.5 0.28 1.310 9.5 0.22 2.120 9.4 0.35 2.430 26 1.51 2.7

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O

OO

O

O

OOH

O

O

HN O

O

OH

O

O

NH

N

O

Cl

fenoxycarb fenoxycarb hemisuccinate

2-(4-phenoxyphenoxy)ethanol chloroxuron

O

O

O

N

Cl

Cl

cypermethrin

Cl Cl

CCl3

p,p′-DDT

O

OOH

O

Cl

Cl

O

NH

N

O

O

diclofop difenoxuron

O

O

O

Cl

ClO

O O N

permethrin pyriproxyfen

even if less marked (33%), also for the 2-(4-fenoxyfenoxy)-ethanol used to synthesise the hapten.

Optimized calibration curve

On the basis of the obtained results, a calibration curve wasoptimized using a 0.1 mol l21 phosphate–citrate buffer, pH 7.4,with 0.2 mol l21 sodium chloride and 20% v/v of methanol. Thecurve has a minimum detection limit of 0.42 ng ml21, with I50of 9.5 ng ml21.

Conclusions

The polyclonal anti-fenoxycarb antiserum used appears to becharacterized by a very high degree of specificity, makingpossible selective determinations of this pesticide by im-munoassay in samples containing other related pesticides orchemicals. The experimental parameters considered (pH, ionicstrength, methanol in buffer) are shown to have an importantinfluence on the calibration curve. It is clear that it is possible toconsider the direct determination of fenoxycarb on real samplesby means of extracting buffers containing limited amounts ofmethanol. At the present time, we are applying this assay tosamples of apple, pear and mulberry leaves, evaluating theeffect of real matrix on the sensitivity and reproducibility of thecalibration curve.

References

1 Pesticide Manual, ed. Worthing, C. R., and Hance, R. J., British CropProtection Council, Farnham, Surrey, United Kingdom, 9th edn.,1991, p. 375.

2 Haenni, R. P., and Mueller, P. A., in Analytical Methods for Pesticideand Plant Growth Regulators, Academic Press, New York, USA,1988, vol. XVI, p. 21.

3 Plantevin, G., Grenier, S., and Chavancy, G., C.R. Acad. Sci. Paris.,1991, 313, 513.

4 Arzone, A., Dolci, M., Marletto, F., and Minero, C., Biosc. Biotech.Biochem., 1995, 59(7), 1318.

5 Hammock, B. D., and Gee, S. J., in Immunoanalysis of Agrochem-icals, ed. Nelson, J. O., Karu, A. E., and Wong, R. B., AmericanChemical Society, Washington, DC, USA, 1995, p. 1.

6 Smith, R. A., and Hiederl, J. B., J. Am. Chem. Soc., 1931, 53, 806.7 Hofle, G., Steglich, W., and Vorbruggen, H., Angew. Chem. Int. Ed.

Engl., 1978, 17, 569.8 Giraudi, G., Baggiani, C., and Giovannoli, C., Anal. Chim. Acta,

1997, 337, 93.9 Bradford, M. M., Anal. Biochem., 1976, 72, 248.

10 Shimizu, F., Kist, M., and Vogt, A., Res. Exp. Med., 1978, 172,231.

11 Giraudi, G., Baggiani, C., Cosmaro, A., Santia, E., and Vanni, A.,Fresenius J. Anal. Chem., 1998, 360, 235.

12 Abraham, G. E., J. Clin. Endocrinol. Metab., 1969, 29, 866

Paper 8/02782JReceived April 14, 1998

Accepted May 6, 1998

Fig. 1 Molecular structures of fenoxycarb and other related chemicals considered in this work.

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