ANALYTICA CHIMICA ACTA

ELSEVIER Analytica Chimica Acta 347 (I 997) 103-l IO

A monoclonal immunoassay for carbofuran and its application to the analysis of fruit juices

Antonio Abad, Maria J. Moreno, Angel Montoya*

L.&oratorio Integmdo de Bioingenieria, Universidad P&t&mica de Valencia, Camino dr Vera s/n., 46022-Wrn&, Spin

Received 25 September 1996; received in revised form 14 January 1997: accepted 24 January 1997

Abstract

Monoclonal antibodies (MAbs) for the carbamate pesticide carbofuran (2,3-dihydro-2 ,2-dimethyl-7-benzofuranyl

methylcarbamate) were obtained by immunizing mice with the hapten 3-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy) carbonyllamino] propanoic acid (BFNP) conjugated to bovine serum albumin (BSA). Based on one of these MAbs in combination with an heterologous hapten, an indirect enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of carbofuran. For standards, the detection limit of the ELISA was 0.2 ng ml-‘, with a lso value of 1.8 ng ml ’ Cross-reactivity studies showed that the immunoassay was quite specific for carbofuran since, of the six N-methylcarbamates assayed, only bendiocarb was significantly recognized (17.4%). The ELISA was applied to the determination of carbofuran in apple, grape, and pineapple juices. Recovery and precision of the method were evaluated by spiking juices with carbofuran at 25, 50, and 100 ng ml-~‘. Coefficients of variation were below 15% in most cases, and mean recoveries were 1 12.6, 107.4, and 1 15.4% for apple, grape, and pineapple juices, respectively. Therefore, the developed ELISA was able to determine carbofuran

at levels below the maximum residue limits simply by diluting the sample. without the need for cleanup or sample concentration.

Kewords: Immunoassay; ELISA; Food analysis; Fruit juices; Carbofuran; N-methylcarbamates

1. Introduction

Carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofur- any1 methylcarbamate) is a widely used systemic and contact N-methylcarbamate pesticide applied to con-

trol insect and nematode pests on a variety of agri- cultural crops. Carbofuran is a cholinesterase inhibitor (IC50 in rats 1.2-3.3x 10P8 M) and it is highly toxic to humans and wildlife through the oral and nasal routes

*Corresponding author. Tel.: +34 6 3877093; fax: +34 6 3877093; e-mail: amontoya@upvnet.upv.es.

of exposure (acute oral LDso in rats X mg kg~- ’ ) [ 11. Like the rest of N-methylcarbamates, the preferred analytical method for carbofuran is liquid chromato- graphy (LC) with post-column derivatization and fluorescence detection [2]. This procedure, although sensitive, is expensive and time-consuming and requires specialized instrumentation. As a conse-

quence of the current public concern about the pre- sence of pesticide residues in water, soil, and food,

there is a growing demand for increased analytical capability and capacity of the laboratories involved in pesticide residue monitoring. In this respect, enzyme

0003-2670/97/$17.00 1‘; 1997 Elsevier Science B.V. All rights reserved.

PII SOOO3-2670(97)00077-9

104 A. Abad et al./Analytica Chimica Acta 347 (1997) 103-110

immunoassays are gaining importance as analytical techniques in the agrochemical field, since they pro-

vide the analytical chemists with a rapid, sensitive, and cost-effective alternative to traditional methods.

Recently, commercial enzyme-linked immunosor- bent assay (ELISA) kits for detecting residues of carbofuran have become available from Ohmicron

and Immunosystems/Millipore (now from Strategic Diagnostics). These immunoassays use polyclonal

antibodies as the specific immunoreagent and they have been applied to the determination of carbofuran in water and soil [3] and in meat and liver [4,5]. Moreover, immunoaffinity columns to purify the pes-

ticide from complex matrices such as potatoes have been prepared from a commercial rabbit polyclonal antiserum [6].

Within a more general objective, consisting of the development of a multianalyte immunoassay for the

most relevant N-methylcarbamate pesticides, we undertook the production of monoclonal antibodies

(MAbs) to carbofuran and the subsequent develop- ment of an ELISA for this insecticide. The carbofuran hapten used to obtain the antibodies was synthesized following the same chemical approach previously

developed in our laboratory to obtain high-affinity MAbs to carbaryl, another N-methylcarbamate insec- ticide [7]. Additional haptens with differences in the

nucleus structure and in the type and length of the spacer arm were also synthesized in order to be tested

as heterologous coating haptens. The most sensitive immunoassay that arose from the different combina-

tions of assay hapten and MAb was evaluated for cross-reactivity to other N-methylcarbamates, and finally applied to the determination of carbofuran in

fruit juices as model matrices.

2. Experimental

2.1. Chemicals and instruments

Analytical pesticide standards were obtained from

Dr. Ehrenstorfer (Augsburg, Germany). Stock solu- tions were prepared in NJ!-dimethylformamide (DMF) and stored at 4°C. Phosgene (20% solution in toluene) was obtained from Fluka (Madrid, Spain). Bovine serum albumin (BSA), ovalbumin (OVA), Tween 20, o-phenylenediamine (OPD), and Freund’s

complete and incomplete adjuvants were purchased from Sigma-Aldrich (Madrid, Spain). Peroxidase- labelled rabbit anti-mouse immunoglobulins were

purchased from Dako (Glostrup, Denmark). Culture plasticware was from Bibby Sterilin (Stone, UK). P3-

X63-Ag8.653 mouse plasmacytoma line was from American Tissue Type Culture Collection (Rockville, MD). Cell culture media (Dulbecco’s Modified Eagle’s Medium, DMEM), foetal calf serum, and

supplements were obtained from Gibco BRL (Paisley, Scotland). All other chemicals and organic solvents were of reagent grade or better.

Polystyrene ELISA plates were from Costar, Cam- bridge, USA (High Binding Plates, cat. no. 3590). ELISA plates were washed with an Ultrawash II microplate washer, and absorbances were read in

dual-wavelength mode (490-630 nm) with a MR 700 microplate reader, both from Dynatech

(Sussex, UK). ‘H nuclear magnetic resonance (NMR) spectra were obtained with a Varian VXR- 400s (400 MHz) spectrometer. Chemical shifts are

given relative to TMS (tetramethylsilane) as an inter- nal reference.

2.2. Hupten synthesis

The structures of the haptens used in this work are

depicted in Fig. 1. BFNP was used as the immunizing hapten to obtain the MAbs, whereas BDNH was used

as heterologous hapten to prepare the coating con- jugate.

CABBOPUBAN (andyte)

BPNP BDNE

(immunhing hapten) (coating hapten)

Fig. 1. Structures of carbofuran and synthesized haptens.

A. Abad et al./Analytica Chimica Acta 347 (1997) 103-110 105

2.2.1. Synthesis of 3-[[(2,3-dihydro-2,2-dimethyl-7- 2.2.2. Synthesis of 6-[[(2,2-dimethyl-1,3-

benzofuranyloxy)carbonyl]amino] propanoic

acid (BFNP) The intermediate compound 2,3-dihydro-2,2-

benzodioxol-4-oxy)carbonyl]amino]hexanoic

acid (BDNH) This compound was synthesized essentially as

dimethyl-7-benzofuranyl chloroformate was first

obtained following a well-known reaction to prepare

chloroformates from aromatic alcohols and phosgene [7,8]. Briefly, 4.54 ml of 2,3-dihydro-2,2-dimethyl-7-

benzofuranol (30.4 mmol) was added to 26 ml of 2.5 M sodium hydroxide. Thereafter, a slight excess of phosgene (25 ml of a 20% phosgene solution in

toluene, 48.3 mmol) was slowly added, and the reac- tion incubated at room temperature for 4 h with mag- netic stirring (WARNING: phosgene is a highly toxic gas. Work in a well ventilated fume hood and handle

carefully). After adding water and methylene chloride, the organic phase was dried over anhydrous sodium

sulphate and evaporated to dryness at reduced pres- sure. On the basis of its behaviour on GC, the 88.9% of the resulting brown solid was assumed to be 2,3-

dihydro-2,2-dimethyl-7-benzofuranyl chloroformate (5.14 g), and subsequently used without further pur-

ification.

described for BFNP, except that 2,2-dimethyl- 1,3- benzodioxol4-ol was used as the starting phenol,

and 6-aminohexanoic acid was used as spacer arm instead of 3-aminopropanoic acid. ‘H NMR (acetone-

d6) 6 1.44 (m, 2 H, CH*), 1.61 (m, 4 H, 2 CH& 1.63 (s,

6 H, 2 CHa), 2.30 (t, 2 H, CH$OOH), 3.20 (m, 2 H, CH2NH), 6.58-6.76 (m, 3 H, aromatic CcHrr).

2.3. Preparation of the immunizing cwjugate

Aminopropanoic acid (2.0 g, 22.6 mmol) was dis- solved in 4 ml of 4 M sodium hydroxide and the solution was cooled at 4°C. 2.57 g of the crude

chloroformate (11.3 mmol) was dissolved in 4 ml of cold 1,6dioxane, and this solution, along with 6 ml of cold 4 M sodium hydroxide, was added to the ami-

noacid solution in five equal portions, with at least

5 min being allowed between additions. The reaction mixture was stirred in an ice bath for 1.5 h. After acidification to pH 4.0 with concentrated hydrochloric acid, the carboxylic derivative, which separated as an

oil, was extracted with ethyl acetate (three 35 ml portions). The ethyl acetate phase was washed several times with diluted hydrochloric acid and extracted

with 1 M bicarbonate solution (three 50 ml portions). After the solution had been cooled in an ice-bath, it was acidified again with concentrated hydrochloric

acid. The precipitated product was collected, washed with water, and dried to yield 1.4 g of

crude BFNl? It was then crystallized from hexane- ethyl acetate (70 : 30) to yield 313.6 mg of pure hapten. ‘H NMR (acetone-dh) 6 1.43 (s, 6 H, 2

CH& 2.62 (t, 2 H, CH&OOH), 3.05 (s, 2 H, CH& 3.45 (q, 2 H. CH*NH), 6.72-6.99 (m, 3 H, aromatic

C6W.

BFNP hapten was covalently attached to BSA using

the modified active ester method 191. 25 pmol of the hapten was incubated overnight at room temperature with stoichiometric amounts of N-hydroxysuccini-

mide and dicyclohexylcarbodiimide in 0.5 ml of

DMF. After centrifuging, 400 pl of the clear super- natant containing the active ester was slowly added to

2 ml of a 15 mg ml-’ BSA solution in 50 mM carbo- nate buffer, pH 9.6. The mixture was allowed to react at room temperature for 4 h with stirring, and finally

the conjugate was purified by gel filtration on Sepha- dex G-50 using 100 mM sodium phosphate buffer, pH

7.4, as eluant. The extent of coupling of the hapten to BSA was determined by UV spectrophotometry. By assuming additive absorbance values, the hapten to

protein molar ratio was evaluated as 19.

2.4. Preparation of the coating conjugate

BDNH hapten was covalently attached to OVA using the mixed-anhydride method [lo]. 18 pmol of the hapten was allowed to react at room temperature for 1 h with stoichiometric amounts of tri-n-butyla-

mine and isobutyl chloroformate in 200 ~1 of DMF. 100 pl of the resulting activated hapten was added to 30 mg of ovalbumin in 2 ml of 50 mM

carbonate buffer, pH 9.6. The coupling reaction was incubated at room temperature for 2 h with stirring, and the obtained conjugate was purified as described

for the immunogen. The extent of coupling of the hapten to OVA was determined by UV spectro- photometry. By assuming additive absorbance values, the hapten to protein molar ratio was evaluated as 6.

106 A. Abad et al./Analyiica Chimica Acta 347 (1997) 103-110

2.5. Production of monoclonal antibodies to carbofuran

Two BALB/c female mice (8-10 weeks old) were immunized with the BSA-BFNP conjugate. Mice

were injected intraperitoneally with 200 1.11 of a 1 : 1 (v/v) mixture of Freund’s complete adjuvant

and PBS (10 mM sodium phosphate, 137 mM NaCl, 2.7 mM KCl, pH 7.4) containing 30 pg of immuno- gen. Two subsequent injections were given at 3

week intervals using incomplete Freund’s adjuvant. Four days before cell fusions, mice were boosted intraperitoneally with 100 pg of immunogen in

200 pl of PBS. The procedure for cell fusion and hybridoma production was based on a modification of the original Kohler and Milstein method [ 11,121.

Details were previously published [7]. Briefly,

mouse spleen cells were fused with the murine mye- loma cell line P3-X63-Ag8.653 using polyethylene-

glycol- 1500 and dispensed into 96-well microtiter culture plates at a cellular density of 4-5 x lo5 cells/ well. Hybridomas were selected in HAT medium, and 12 days after fusion cell culture supernatants were

tested for the presence of anti-carbofuran antibodies by a simultaneous non-competitive/competitive

ELISA. Wells that gave a strong positive response

in the non-competitive ELISA (absorbance 2 1 .O) and that showed a very significant carbofuran recognition (usually a signal reduction higher than 80% in the

competitive format with 1 pM carbofuran) were cloned by limiting dilution to ensure monoclonality. Clones were expanded and stored in liquid nitrogen, and cell culture supernatants containing the MAbs

were stored at 4°C and used without further purifica- tion.

2.6. Preparation of carbofuran standards and samples

From a 100 pM stock solution of carbofuran in

DMF, standards in the range 0.2 pg ml-’ to 880 ng ml-’ were prepared daily by serial dilution

in 2xPBS. Fruit juices (100% pure) were commercial samples

obtained from local supermarkets and they were used without any sample pre-treatment. For ELISA deter- mination, spiked juice samples were diluted l/l0 and

1125 in 2 xPBS and each dilution was analyzed in

triplicate. The carbofuran concentration in the samples

was calculated by averaging values obtained at each dilution.

2.7. Carbofuran immunoassay

A competitive indirect heterologous ELISA format was chosen. Immunoassays were performed by the following procedure:

96-well microtiter plates were coated with the OVA-BDNH conjugate (1 pg ml-‘, 100 @/well) in coating buffer (50 mM carbonate-bicarbonate buffer,

pH 9.6) by overnight incubation at room temperature. Plates were washed four times with washing solution

(0.15 M NaCl containing 0.05% Tween 20), and 50 pl/ well of standards (in quadruplicate) or samples (in triplicate) were added, followed by 50 pi/well of a l/

400 dilution of the LIB-BFNP23 MAb in 2xPBS containing 0.1% BSA. After incubation at room tem- perature for 2 h, the plates were washed as before, and 100 pl of a l/1000 dilution of peroxidase-labelled rabbit anti-mouse immunoglobulins in PBS contain-

ing 0.05% Tween 20 was added to the wells. Plates were incubated at room temperature for 1 h. and after

another washing step, 100 ul of a 2 mg ml-’ OPD solution in reaction buffer (25 mM citrate, 62 mM

sodium phosphate, pH 5.4, containing 0.012% HzOz) was added. After 10 min at room temperature, the enzymic reaction was stopped by adding 100 pl of 2.5 M sulfuric acid, and the absorbance at 490 nm was read.

Using a commercial software package (Sigmaplot, Jandel Scientific), absorbance values from standards were mathematically fitted to a four-parameter logistic equation:

y = {(A - D)/[l + (x/C)“““‘~]} + D,

where A is the asymptotic maximum, B is the curve slope at the inflexion point, C is the x value at the inflexion point (corresponding to the analyte concen- tration that reduces absorbance to 50% of the max- imum, [so), and D is the asymptotic minimum. Carbofuran concentration of samples was determined by interpolation of the mean absorbance on the result- ing standard curve.

A. Ahad et al. /Analytica Chimica Acta 347 (1997) 103-l 10 107

3. Results and discussion Table 1

Cross-reactivity a of several compounds with LIB-BFNP23 MAb

3.1. Immunoassay development

The first and most critical step in the development of immunoassays for pesticides is the synthesis of pesticide-like molecules containing a functional group suitable for protein conjugation. The hapten used to

obtain the MAbs (BFNP) was designed as a result of previous studies carried out in our laboratory with

carbaryl, a pesticide that also belongs to the iv-methyl- carbamate family. The synthesis strategy essentially

consisted of the introduction of a carboxylic spacer arm through the methyl group of the carbamate moi-

ety. From two cell fusion experiments with spleno- cytes from mice immunized with a BSA conjugate of this hapten, two hibridomas secreting antibodies that competitively bound free (unconjugated) carbofuran at the nanomolar level were identified.

Compound

Carbofuran

2,3-Dihydro-

2,2-dimethyl-

7-benzofuranol

Bendiocarb

Carbaryl

A valuable approach to improve the sensitivity of

pesticide immunoassays is the use of assay conjugates bearing haptens different from that employed for antibody production (heterologous haptens). There-

fore, we synthesized 11 heterologous haptens differing from BFNP in the heterocyclic nucleus and/or in the

length and type of the spacer arm (not shown). Com- petitive curves for carbofuran were obtained with the two selected MAbs using coating conjugates of these new haptens, and a 4-fold improvement in ELISA sensitivity was achieved with some of these hetero- logous conjugates. The most sensitive immunoassay was found to be that based on the MAb designated LIB-BFNP23, together with the heterologous conju-

gate derived from the BDNH hapten, prepared as described in Section 2. This combination of immu- noreagents was subsequently used for further work.

Methiocarb

Propoxur

Aldicarb

Methomyl

3.2. Specificity of the carbofuran immunoassay

The ability of LIB-BFNP23 MAb to recognize the compounds listed on Table 1 was tested by performing competitive assays and determining their respective

15, (nM) values. The immunoassay proved to be very specific for carbofuran, since most of the assayed N- methylcarbamate pesticides were not significantly recognized (<I%). Only bendiocarb, a scarcely used pesticide very similar in structure to carbofuran, was recognized at a significant extent (17.4%). It is inter-

a Cross-reactivity was calculated as

(

150 carbofuran

1x0 compound > X 100.

I,, for carbofuran wa\ 8. I nM

esting to point out that the N-methylcarbamate group of carbofuran is a very important antigenic determi- nant, as judged by the loss of recognition found when it is not present (2.3% cross-reactivity of 2,3-dihydro- 2,2-dimethyl-7-benzofuranol). This result confirmed our previous finding obtained with anti-carbaryl MAbs 171.

Chemical

structure

pcoNHcHl

Cross-reactivity

(54,

0.60

OCONHCII, 0.60

108 A. Abad et al./Analytica Chimica Acta 347 (1997) 103-110

10-d lo-3 10-z 10-l 100 10' 102 103

[CARBOFURAN] (ng mr’)

Fig. 2. Normalized average standard curve for carbofuran ELISA.

Each point represents the mean of 24 value&standard deviation.

3.3. Analytical characteristics of the ELISA for carbofuran

A standard curve for the carbofuran competitive immunoassay is shown in Fig. 2. This sigmoidal curve was obtained by averaging 24 individual stan-

dard curves performed in the course of carbofuran determination in juice samples. In order to be

properly averaged, individual curves were

normalized by expressing the absorbance (A4& as

the percentage of the maximum response ( 100xA4&

A 490,max). In the dynamic range of the curve, signal

coefficients of variation were between 6.3%

(0.22 ng ml-‘) and 14.8% (13.8 ng ml-‘). Table 2 illustrates the variability of the mathematical para- meters that define the sigmoidal equation. Data

were calculated from the individual standard curves fitted without normalization, as used for direct inter- polation of samples. The mean carbofuran Iso was 1.8 ng ml-’ and the detection limit, estimated as the carbofuran concentration that reduced

absorbance to 90% of the maximum, was 0.2 ng ml-‘. Both parameters indicate that this ELISA can be considered very sensitive for carbofuran determina- tion.

Table 2

Variability of the mathematical parameters of the sigmoidal

standard curve

Parameter a

A B C (150) D

Mean b 1.134 2.599 1.847 0.025

SD. 0.222 0.218 0.432 0.010

C.V. (%) 19.58 8.39 23.39 40.00

Minimum 0.681 2.243 1.286 0.003

Maximum 1 SO7 2.979 2.897 0.046

a Asymptotic maximum absorbance (A), slope at the inflexion point

(B), concentration value in ng ml-’ at the inflexion point (C), and

asymptotic minimum absorbance (D) are the parameters which fit

;h;22;ydal equation y = {(A - D)/[I + (x/C)“‘“‘~]} + D.

3.4. Analysis of spiked juice samples

Since food matrix effects are quite common in immunoassays, the influence of the juices of interest on the ELISA was roughly tested prior to the applica-

tion of the method to samples spiked with the analyte. Carbofuran standards were prepared in quadruplicate in 2 xPBS containing a variable proportion of each juice, and the matrix influence on the parameters of the standard curve was analyzed. At matrix proportions

lower than l/10, the curve slope, Adgo,__, and Z5a varied less than 10%. However, at matrix proportions

higher than l/l0 the curve parameters were appreci- ably modified (variations higher than 20%). These results suggested that carbofuran could be analyzed in

juices simply by diluting samples l/10-1/20 in

2xPBS, without the need of a cleanup step. Apple, grape, and pineapple juices were spiked with

carbofuran at 25, 50, and 100 ng ml-‘, and directly analyzed by ELISA without any pre-treatment other

than dilution. Each ELISA plate included its own carbofuran standard curve, and absorbances from samples were interpolated on the curve performed in the same plate. Table 3 shows the results obtained for each fortified juice sample determined 10 times at both l/10 and l/25 dilutions (l/20 and l/50 in the assay, respectively). Precision obtained for all fruit juices can be considered very good for a residue method, since most coefficients of variation were

around or below 15%. Recoveries were also good (mean 112.6%, 107.4%, and 115.4% for apple, grape, and pineapple juices, respectively), although the

A. Abad et al. /Analytica Chimica Acta 347 (1997) 103-I 10 I09

Table 3

Reproducibility and accuracy of the carbofuran ELISA for spiked juice samples

Carbofuran spiked level (ng ml-‘)

Apple juice Grape juice Pineapple juice

25 50 100 25 50 100 25 50 100 Mean value found ” 28.14 58.50 108.21 25.06 54.16 112.44 26.84 60.80 117.32 S.D. 4.23 7.74 9.06 5.63 9.18 14.31 6.70 9.64 14.07 C.V. (%) 15.02 13.23 8.37 22.47 16.77 12.72 26.05 15.X6 12.00 Minimum 2 1.90 43.38 92.95 13.39 33.85 89.09 12.06 46.68 97.00 Maximum 37.55 74.08 121.42 36.68 72.85 140.50 36.39 75.15 144.93 Recovery (%) I 12.56 117.00 108.21 100.24 109.52 112.44 107.36 121.60 117.32

a Data are the average of 10 independent determinations. Each determination consisted of the mean of the carbofurdn values obtained at l/20

and l/50 dilutions.

immunoassay showed a slight tendency to overesti- mate. This could be indicating that matrix effects were not completely eliminated at the dilutions used in the assay. Juice samples without carbofuran (non-spiked samples) were also routinately included in the analysis

as negative controls. Carbofuran concentrations below

the detection limit of the assay were found in all cases, so no false positive results were obtained.

4. Conclusions

To our knowledge, the MAbs for carbofuran herein described are the first reported for this pesticide. With

one of the MAbs, a heterologous indirect ELISA with a Z50 for standards of 1.8 ng ml-’ was developed. The sensitivity of this immunoassay is only slightly lower

than that of the magnetic particle-based ELISA devel- oped by Jourdan et al. [ 31 and commercially available from Strategic Diagnostics (Zs0=0.8 ng ml-‘). Never-

theless, the fact that our immunoassay uses MAbs can

be a potential advantage since MAbs constitute a homogeneous, perfectly standardized immunorea-

gent. The monoclonal ELISA presented here is able to

determine down to 25 ng ml-’ of carbofuran in apple, grape, and pineapple juices. No previous application of an immunoassay to the determination of carbofuran

in fruit juices has been reported. In fact, to date very few immunoassays have been applied to pesticide determination in fruit juices, despite the fact that they constitute an excellent model to evaluate the suitabil- ity of ELISAs for the analysis of residues in more complex and relevant matrices, such as whole fruits

and vegetables. The method has proved to be very robust, since samples can be analyzed simply by dilution, without any concentration or cleanup step. The simplicity of the method is clearly evidenced by

its sample throughput. In the described conditions, with a standard curve included in quadruplicate, 16

samples can be determined in triplicate on each plate.

ELISA takes less than 4 h to be performed, and four plates can be easily handled simultaneously. By over- lapping incubation times, the analyst can process eight

plates in a working day, so as many as 120 juice samples can be determined daily.

Although perfectly adequate for the analysis

of fruits and vegetables, the sensitivity of this

ELISA is not sufficient to determine carbofuran in drinking water at the low levels established by the European legislation (0.1 ng ml- I). Therefore, the influence of several physicochemical factors and

ELISA format is currently under study to optimize the assay and to improve its sensitivity. Further work is

also in progress to perform new cell fusions searching

for carbofuran antibodies with higher affinity and specificity.

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