Hapten Synthesis and Production of Monoclonal Antibodies to the N-Methylcarbamate Pesticide Methiocarb

Article abstract of DOI:10.1021/jf980077v

With the aim of developing an immunoassay for the detection of the insecticide methiocarb, three compounds with carboxylic spacer arms of different lengths introduced at the carbamate group were synthesized, conjugated to proteins, and used as immunizing haptens in mice. Monoclonal antibodies (MAbs) were subsequently obtained and characterized for their affinity to methiocarb. In the homologous conjugate-coated assay format, MAbs exhibited I_5o values in the 1 -10 nM range. Thereafter, a collection of methiocarb haptens with modifications in the aromatic ring structure or in the spacer arm were synthesized to be used as heterologous haptens. These haptens and MAbs were incorporated into several ELISA configurations. Some of the new combinations of immunoreagents and assay format provided a significant improvement in assay sensitivity, reaching I₅₀ values around 0.1 nM. MAbs seem to be very specific for methiocarb, as evidenced by the negligible cross-reactivity shown by methiocarb metabolites. These immunoreagents are very promising analytical tools for the rapid and sensitive determination of methiocarb in food and in the environment.

Full text of DOI:10.1021/jf980077v

J. Agric. Food Chem. 1998, 46, 2417−2426
2417
Ha p ten Syn th esis a n d P r od u ction of Mon oclon a l An tibod ies to th e
N-Meth ylca r ba m a te P esticid e Meth ioca r b
Antonio Abad, Mar´ıa J ose´ Moreno, and Angel Montoya*
Laboratorio Integrado de Bioingenier´ıa, Universidad Polite´cnica de Valencia, Camino de Vera s/n,
46022 Valencia, Spain
With the aim of developing an immunoassay for the detection of the insecticide methiocarb, three
compounds with carboxylic spacer arms of different lengths introduced at the carbamate group were
synthesized, conjugated to proteins, and used as immunizing haptens in mice. Monoclonal antibodies
(MAbs) were subsequently obtained and characterized for their affinity to methiocarb. In the
homologous conjugate-coated assay format, MAbs exhibited I50 values in the 1-10 nM range.
Thereafter, a collection of methiocarb haptens with modifications in the aromatic ring structure or
in the spacer arm were synthesized to be used as heterologous haptens. These haptens and MAbs
were incorporated into several ELISA configurations. Some of the new combinations of immuno-
reagents and assay format provided a significant improvement in assay sensitivity, reaching I50
values around 0.1 nM. MAbs seem to be very specific for methiocarb, as evidenced by the negligible
cross-reactivity shown by methiocarb metabolites. These immunoreagents are very promising
analytical tools for the rapid and sensitive determination of methiocarb in food and in the
environment.
Keyw or d s: ELISA; immunoassay; insecticide; monoclonal antibodies; hapten design; hapten
heterology; ELISA format; carbamate
INTRODUCTION
than for the highly sensitive routine analysis of car-
bamate residues. Based on the pioneering work by
Moye et al. (1977), further modified for use on food
samples by Krause (1985), HPLC using postcolumn
derivatization and fluorescence detection (EPA method
531.1) is the preferred technique for the sensitive and
selective determination of N-methylcarbamate pesti-
cides in environmental waters and food (McGarvey,
1993; Yang et al., 1996). However, the method presents
some limitations derived from the fact that laborious
cleanup, concentration, and derivatization steps are
needed to obtain the desired sensitivity. Thus, the
method requires sophisticated equipment that is not
available in most analytical laboratories, and it is not
very well suited for the analysis of a large number of
samples. In fact, most pesticide regulatory programs
analyze for N-methylcarbamates only a part of the total
number of food samples analyzed for other active
ingredients, such as organophosphorus pesticides (Gun-
derson, 1995).
Immunoassays are analytical methods based on the
interaction of an analyte with an antibody that recog-
nizes it with high affinity and specificity. They are
simple, cost-effective, and field-portable, do not require
sophisticated instrumentation, and are able to analyze
a large number of samples simultaneously. All of these
features, along with the large number of pesticides for
which antibodies have been produced over the past few
years, have promoted the acceptance of immunochemi-
cal techniques among analytical chemists as alternative
and/or complementary methods for the analysis of
agrochemicals (Hammock and Gee, 1995; Meulenberg
et al., 1995; Dankwardt and Hock, 1997). This is
particularly true for pesticides that are difficult and/or
costly to determine due to their physicochemical char-
Methiocarb [4-(methylthio)-3,5-xylyl methylcarbam-
ate], also known as mercaptodimethur or mesurol, is a
nonsystemic N-methylcarbamate insecticide and acari-
cide with contact and stomach action that is particularly
effective for control of mites, including organophosphate-
resistant strains. Likewise, methiocarb is a mollusci-
cide with neurotoxic action that has been employed to
control slugs and snails in a wide range of agricultural
situations, and it has also been used as a bird repellent
on blueberries, cherries, and grapes (Worthing and
Hance, 1991; Dolbeer et al., 1994). Methiocarb hydro-
lyzes quite rapidly in water under natural sunlight to
afford 4-(methylthio)-3,5-xylenol (Chiron et al., 1996).
In plants, methiocarb is metabolized to its sulfoxide and
sulfone derivatives (Miller et al., 1985). Like the parent
compound, methiocarb sulfoxide and methiocarb sulfone
are cholinesterase inhibitors.
Methiocarb, like most N-methylcarbamates, is ther-
mally unstable and decomposes to the phenol under the
usual gas chromatography conditions (Lisˇka and Slo-
bodn´ık, 1996). This problem can be overcome by vary-
ing the operating conditions (Mueller and Stan, 1990)
or by derivatization of the carbamate pesticides (Ball-
esteros et al., 1993; Fa¨rber and Scho¨ler, 1993; Bakowski
et al., 1994). Several reports have been recently pub-
lished dealing with the coupling of mass spectrometry
with liquid chromatography for the determination of
N-methylcarbamates (Chiron et al., 1996; Di Corcia et
al., 1996; Slovodnik et al., 1997), but these methods
seem to be more appropriate for confirmation purposes
* Author to whom correspondence should be addressed
(telephone 34-96-3877093; fax 34-96-3877093; e-mail
amontoya@eln.upv.es).
S0021-8561(98)00077-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/23/1998

2418 J. Agric. Food Chem., Vol. 46, No. 6, 1998
Abad et al.
F igu r e 1. Reaction scheme of the synthesis of hapten series MXN*, MCN*, DPN*, MPN*, and PN*.
solutions were prepared in dry N,N-dimethylformamide (DMF)
and stored at 4 °C. Starting products for the synthesis of
haptens and hapten-protein coupling reagents were obtained
from Fluka-Aldrich Qu´ımica (Madrid, Spain).
acteristics or for large monitoring programs. In these
situations the use of immunoassays as a screening
technique could provide a great saving of resources,
effort, and toxic solvents, since fortunately most samples
do not contain residues or they contain them below
violative levels. Therefore, although immunoassays are
not the panacea, there is nowadays little doubt that they
constitute a sensitive, selective, and inexpensive method
for the rapid analysis of pesticide residues in a variety
of matrixes.
The aim of this work was to obtain high-affinity
monoclonal antibodies (MAbs) to methiocarb and to
develop enzyme immunoassays as an alternative to
liquid chromatographic techniques for the screening of
large numbers of agricultural and environmental
samples. The achievement of this objective strongly
relies on a proper hapten design since the structure of
the immunizing hapten is primarily responsible for the
antibody recognition properties. Moreover, assay sen-
sitivity is often influenced by the structure of the assay
hapten (Harrison et al., 1991a; Goodrow et al., 1995;
Szurdoki et al., 1995). Accordingly, a high number of
haptens with different spacer arms and with modifica-
tions in the aromatic moiety of the methiocarb structure
were synthesized and conjugated to carrier proteins and
to horseradish peroxidase (HRP). The characterization
of the MAbs for sensitivity and specificity is presented,
and the influence of heterologous haptens in different
assay formats is evaluated for the development of
immunoassays to this pesticide.
Ovalbumin (OVA), Freund’s adjuvants, and o-phenylenedi-
amine (OPD) were obtained from Sigma Qu´ımica (Madrid,
Spain). Bovine serum albumin fraction V (BSA), enzyme
immunoassay grade HRP, and poly(ethylene glycol) (PEG)
1500 were purchased from Boehringer Mannheim (Barcelona,
Spain). Peroxidase-labeled rabbit anti-mouse immunoglobu-
lins and affinity-isolated goat anti-mouse immunoglobulins
were obtained from Dako (Glostrup, Denmark). Culture media
(Dulbecco’s Modified Eagle’s Medium, DMEM), fetal calf serum
(Myoclone Super Plus), and supplements were from GibcoBRL
(Paisley, Scotland). Culture plasticware was from Bibby
Sterilin Ltd. (Stone, U.K.). P3-X63-Ag8.653 mouse plasma-
cytoma line was from American Tissue Type Culture Collection
(Rockville, MD).
Polystyrene culture plates (High Binding Plates, catalog no.
3590) were from Costar (Cambridge, MA). ELISA plates were
washed with an Ultrawash II microplate washer from Dyna-
tech (Sussex, U.K.), and absorbances were read in dual-
wavelength mode (490-650 nm) with a Emax microplate
reader from Molecular Devices (Sunnyvale, CA). 1H and 13C
nuclear magnetic resonance (NMR) spectra were obtained with
a Varian Gemini 300 spectrometer (Sunnyvale, CA), operating
at 300 MHz for 1H and at 75 MHz for 13C. Chemical shifts
are reported relative to tetramethylsilane. Liquid secondary
ion (LSI) (3-nitrobenzyl alcohol as matrix) mass spectra (MS)
were obtained on a VG Autospec mass spectrometer (Kyoto,
J apan). Ultraviolet-visible (UV-vis) spectra were recorded
on a UV-160A Shimadzu spectrophotometer (Kyoto, J apan).
Ha p ten Syn th esis. Methiocarb haptens used in this work
were prepared by introduction of alkyl chain spacers, ending
in a carboxylic acid, at the hydroxyl group of the phenolic
precursor, either after being reacted with phosgene to form
the carbamate group (Figure 1), or directly by O-alkylation
(Figure 2).
MATERIALS AND METHODS
Ch em ica ls, Im m u n or ea gen ts, a n d In str u m en ts. Stand-
ards of methiocarb, methiocarb sulfoxide, and methiocarb
sulfone were from Riedel-de Hae¨n (Seelze, Germany). Stock

Monoclonal Antibodies to Methiocarb
J. Agric. Food Chem., Vol. 46, No. 6, 1998 2419
product as an oil. The product was crystallized from hexane/
ethyl acetate (70:30) to obtain 840 mg of the pure hapten
(22.9%): 1H NMR (acetone-d6) δ 1.38-1.70 (m, 6 H, CH2-
CH2-CH2), 2.20 (s, 3 H, S-CH3), 2.32 (t, 2 H, CH2-COOH),
2.52 (s, 6 H, 2 CH3), 3.21 (q, 2 H, CH2-NH), 6.90 (s, 2 H,
aromatic); 13C NMR (acetone-d6) δ 18.32, 21.84, 25.30, 26.95,
34.07, 41.52, 121.97, 131.80, 144.55, 152.05, 155.05, 174.63;
LSI-MS, m/z (relative intensity) 325 (90, M), 168 (100).
From appropriate amino acids and phenolic precursors, the
following compounds were also synthesized with similar yields
by using the same procedure.
3-[[1-(4-(Methylthio)-3,5-xylyloxy)carbonyl]amino]propano-
ic Acid (MXNP): 1H NMR (acetone-d6) δ 2.20 (s, 3 H, S-CH3),
2.52 (s, 6 H, 2 CH3), 2.60 (t, 2 H, CH2-COOH), 3.46 (q, 2 H,
CH2-NH), 6.90 (s, 2 H, aromatic); 13C NMR (acetone-d6) δ
18.32, 21.84, 34.45, 37.73, 121.94, 131.86, 144.60, 152.05,
155.05, 173.100; LSI-MS, m/z (relative intensity) 283 (100, M),
168 (95).
4-[[1-(4-(Methylthio)-3,5-xylyloxy)carbonyl]amino]butanoic
Acid (MXNB): 1H NMR (acetone-d6) δ 1.86 (m, 2 H, CH2-CH2-
CH2), 2.20 (s, 3 H, S-CH3), 2.40 (t, 2 H, CH2-COOH), 2.52 (s,
6 H, 2 CH3), 3.26 (q, 2 H, CH2-NH), 6.91 (s, 2 H, aromatic);
13C NMR (acetone-d6) δ 18.33, 21.85, 25.84, 31.37, 41.10,
122.00, 131.86, 144.58, 152.15, 155.20, 174.40; LSI-MS, m/z
(relative intensity) 297 (100, M), 168 (80).
3-[[1-(3,5-xylyloxy)carbonyl]amino]propanoic Acid (DPNP):
1H NMR (acetone-d6) δ 2.25 (s, 6 H, 2 CH3), 2.60 (t, 2 H, CH2-
COOH), 3.45 (q, 2 H, CH2-NH), 6.71-6-81 (m, 3 H, aromatic);
13C NMR (acetone-d6) δ 21.09, 34.44, 37.66, 120.07, 127.12,
139.46, 152.33, 155.33, 173.12.
4-[[1-(3,5-xylyloxy)carbonyl]amino]butanoic Acid (DPNB): 1H
NMR (acetone-d6) δ 1.84 (m, 2 H, CH2-CH2-CH2), 2.25 (s, 6
H, 2 CH3), 2.40 (t, 2 H, CH2-COOH), 3.25 (q, 2 H, CH2-NH),
6.71-6-81 (m, 3 H, aromatic); 13C NMR (acetone-d6) δ 21.14,
25.86, 31.37, 41.03, 120.13, 127.10, 139.45, 152.44, 155.52,
174.43.
6-[[1-(3,5-xylyloxy)carbonyl]amino]hexanoic Acid (DPNH):
1H NMR (acetone-d6) δ 1.35-1.70 (m, 6 H, CH2-CH2-CH2),
2.25 (s, 6 H, 2 CH3), 2.30 (t, 2 H, CH2-COOH), 3.19 (q, 2 H,
CH2-NH), 6.70-6.80 (m, 3 H, aromatic); 13C NMR (acetone-
d6) δ 21.14, 25.28, 26.94, 34.07, 41.49, 120.13, 127.04, 139.43,
152.43, 155.55, 174.66; LSI-MS, m/z (relative intensity) 279
(100, M), 123 (50).
3-[[1-(4-(Methylthio)-3-cresyloxy)carbonyl]amino]propanoic
Acid (MCNP): 1H NMR (acetone-d6) δ 2.27 (s, 3 H, CH3), 2.44
(s, 3 H, S-CH3), 2.60 (t, 2 H, CH2-COOH), 3.45 (q, 2 H, CH2-
NH), 6.94 (m, 2 H, aromatic), 7.17 (m, 1 H, aromatic); 13C NMR
(acetone-d6) δ 15.45, 19.93, 34.42, 37.69, 120.62, 123.87, 126.74,
134.58, 137.39, 149.81, 155.28, 173.10.
4-[[1-(4-(Methylthio)-3-cresyloxy)carbonyl]amino]butanoic Acid
(MCNB): 1H NMR (acetone-d6) δ 1.86 (m, 2 H, CH2-CH2-
CH2), 2.29 (s, 3 H, CH3), 2.41 (t, 2 H, CH2-COOH), 2.46 (s, 3
H, S-CH3), 3.26 (q, 2 H, CH2-NH), 6.96 (m, 2 H, aromatic),
7.20 (m, 1 H, aromatic); 13C NMR (acetone-d6) δ 21.14, 25.86,
31.37, 41.03, 120.13, 127.10, 139.45, 152.44, 155.52, 174.43.
6-[[(1-(4-(Methylthio)-3-cresyloxy)carbonyl]amino]hexanoic
Acid (MCNH): 1H NMR (acetone-d6) δ 1.35-1.70 (m, 6 H,
CH2-CH2-CH2), 2.29 (s, 3 H, CH3), 2.32 (t, 2 H, CH2-COOH),
2.45 (s, 3 H, S-CH3), 3.20 (q, 2 H, CH2-NH), 6.95 (m, 2 H,
aromatic), 7.19 (m, 1 H, aromatic); 13C NMR (acetone-d6) δ
15.59, 19.93, 25.24, 26.91, 34.00, 41.36, 120.68, 123.90, 126.78,
134.40, 137.39, 149.96, 155.28, 174.56; LSI-MS, m/z (relative
intensity) 311 (100, M).
3-[[1-(4-(Methylthio)phenyloxy)carbonyl]amino]propanoic Acid
(MPNP): 1H NMR (acetone-d6) δ 2.47 (s, 3 H, S-CH3), 2.60 (t,
2 H, CH2-COOH), 3.45 (q, 2 H, CH2-NH), 7.08 (m, 2 H,
aromatic), 7.25 (m, 2 H, aromatic); 13C NMR (acetone-d6) δ
16.65, 34.91, 38.20, 123.60, 128.77, 135.95, 150.63, 155.70,
173.60.
4-[[1-(4-(Methylthio)phenyloxy)carbonyl]amino]butanoic Acid
(MPNB): 1H NMR (acetone-d6) δ 1.84 (m, 2 H, CH2-CH2-
CH2), 2.40 (t, 2 H, CH2-COOH), 2.46 (s, 3 H, S-CH3), 3.25
(q, 2 H, CH2-NH), 7.08 (m, 2 H, aromatic), 7.25 (m, 2 H,
aromatic); 13C NMR (acetone-d6) δ 16.69, 26.29, 31.83, 41.56,
123.64, 128.79, 135.85, 150.72, 155.83, 174.91.
F igu r e 2. Reaction scheme of the synthesis of hapten series
MXO*.
6-[[1-(4-(Methylthio)-3,5-xylyloxy)carbonyl]amino]hexano-
ic Acid (MXNH, Figure 1). To a solution of 14.31 g (85.2 mmol)
of 4-(methylthio)-3,5-xylenol in 100 mL of 2.5 M sodium
hydroxide in water was added 50 mL of a 20% phosgene
solution in toluene (96.5 mmol). (WARNING: phosgene is a
highly toxic gas. Work in a well ventilated fume hood and
handle carefully.) The mixture was stirred for 4 h at room
temperature, and the organic phase was evaporated to dryness
at reduced pressure to afford 18.21 g of a yellow oil, 15.48 g of
which was identified as the chloroformate by GC (78.8%). This
crude product was used without further purification in the
following step: 2.96 g of aminohexanoic acid (22.6 mmol) was
dissolved in 4.0 mL of 4 M sodium hydroxide, and the solution
was cooled at 4 °C. 1-[4-(Methylthio)-3,5-xylyl] chloroformate
(3.0 g, 11.3 mmol) was dissolved in 4 mL of 1,4-dioxane and
cooled at 4 °C. The dioxane solution, along with 3.0 mL of
cold (4 °C) 4 M sodium hydroxide, was added to the amino-
hexanoic solution in five equal portions, with ∼5 min being
allowed between additions. The reaction mixture was stirred
in an ice bath for 1.5 h. After acidification to pH 4 with
concentrated hydrochloric acid, the carboxylic derivative was
extracted with ethyl acetate (three 50 mL portions). The ethyl
acetate phase was washed several times with diluted hydro-
chloric acid and extracted with 1 M bicarbonate solution (two
50 mL portions). The aqueous phase was acidified again with
concentrated hydrochloric acid, extracted with ethyl acetate,
and dried over anhydrous sodium sulfate, and the solvent was
evaporated at reduced pressure to afford 1.19 g of the crude

2420 J. Agric. Food Chem., Vol. 46, No. 6, 1998
Abad et al.
6-[[1-(4-(Methylthio)phenyloxy)carbonyl]amino]hexanoic Acid
(MPNH): 1H NMR (acetone-d6) δ 1.35-1.70 (m, 6 H, CH2-
CH2-CH2), 2.30 (t, 2 H, CH2-COOH), 2.46 (s, 3 H, S-CH3),
3.19 (q, 2 H, CH2-NH), 7.08 (m, 2 H, aromatic), 7.25 (m, 2 H,
aromatic); 13C NMR (acetone-d6) δ 16.70, 25.76, 27.40, 30.71,
34.53, 42.00, 123.61, 128.80, 135.77, 150.76, 155.73, 175.14.
3-[[1-(Phenyloxy)carbonyl]amino]propanoic Acid (PNP): 1H
NMR (acetone-d6) δ 2.61 (t, 2 H, CH2-COOH), 3.46 (q, 2 H,
CH2-NH), 7.09-7.37 (m, 5 H, aromatic); 13C NMR (acetone-
d6) δ 34.69, 37.69, 122.46, 125.62, 129.86, 152.45, 155.21,
173.10.
protein, and the corresponding conjugate. By assuming that
the molar absorptivity of haptens was the same for the free
and conjugated forms, apparent molar ratios were estimated
as 27, 16, and 18 for haptens MXNP, MXNB, and MXNH,
respectively.
P r ep a r a tion of Coa tin g Con ju ga tes. All of the haptens
were covalently attached to OVA using the mixed-anhydride
method (Rajkowski et al., 1977). Eighteen micromoles of the
hapten was allowed to react at room temperature for 1 h with
stoichiometric amounts of tri-n-butylamine and isobutyl chlo-
roformate in 200 µL of DMF. One hundred microliters of the
resulting activated hapten was added to 30 mg of OVA in 2
mL of 50 mM carbonate buffer, pH 9.6. The coupling reaction
was incubated at room temperature for 2-3 h with stirring,
and the conjugates obtained were purified as described for the
immunogens. The extent of coupling of each hapten to OVA
was determined by UV spectrophotometry. By assuming
additive absorbance values, hapten to protein molar ratios
were evaluated as 5, 5, 6, 9, 8, 8, 2, 4, 4, 2, 2, 4, 9, 6, 2, 2, 4,
and 6 for haptens MXNP, MXNB, MXNH, DPNP, DPNB,
DPNH, MCNP, MCNB, MCNH, MPNP, MPNB, MPNH, PNP,
PNB, PNH, MXOA, MXOB, and MXOH, respectively.
P r ep a r a tion of En zym e Con ju ga tes. The mixed-anhy-
dride method was also used for covalent coupling of haptens
to HRP. Typically, 2.9 µL of tributylamine and 1.6 µL of
isobutyl chloroformate were added to 13.3 µmol of the hapten
in 200 µL of DMF. The mixture was stirred for 1 h at room
temperature. After the addition of 1.8 mL of DMF, 100 µL of
this diluted solution of activated hapten was incubated for 2
h at room temperature with 1 mL of a 2.2 mg/mL solution of
HRP in 50 mM carbonate buffer, pH 9.6. HRP-hapten
conjugates were purified as described for the immunogens.
HRP conjugate concentrations and molar ratios were estimated
spectrophotometrically. With the same assumptions as before,
the estimated molar ratios were 1.7, 3.0, 4.6, 11.5, 11.3, 11.5,
2.3, 1.4, 3.2, 1.7, 3.4, and 1.5 for haptens MXNP, MXNB,
MXNH, DPNP, DPNB, DPNH, MCNP, MCNB, MCNH, MXOA,
MXOB, and MXOH, respectively.
P r od u ction of Mon oclon a l An tibod ies to Meth ioca r b.
Immunization. BALB/c female mice (8-10 weeks old) were
immunized with BSA-MXNP, -MXNB, and -MXNH conju-
gates. First dose consisted of 30 µg of conjugate intraperito-
neally injected as an emulsion of PBS and complete Freund’s
adjuvant. Two subsequent injections were given at 3-week
intervals emulsified in incomplete Freund’s adjuvant. One
week after the last injection, mice were tail-bled and sera
tested for anti-hapten antibody titer by indirect ELISA and
for analyte recognition properties by competitive indirect
ELISA. After a resting period of at least 3 weeks from the
last injection in adjuvant, mice selected to be spleen donors
for hybridoma production received a final soluble intraperi-
toneal injection of 100 µg of conjugate in PBS, 4 days prior to
cell fusion.
4-[[1-(Phenyloxy)carbonyl]amino]butanoic Acid (PNB): 1H
NMR (acetone-d6) δ 1.88 (m, 2 H, CH2-CH2-CH2), 2.40 (t, 2
H, CH2-COOH), 3.26 (q, 2 H, CH2-NH), 7.09-7.37 (m, 5 H,
aromatic); 13C NMR (acetone-d6) δ 25.80, 31.34, 41.04, 122.50,
125.54, 129.85, 152.52, 155.37, 174.42.
6-[[1-(Phenyloxy)carbonyl]amino]hexanoic Acid (PNH): 1H
NMR (acetone-d6) δ 1.36-1.68 (m, 6 H, CH2-CH2-CH2), 2.30
(t, 2 H, CH2-COOH), 3.20 (q, 2 H, CH2-NH), 7.09-7.37 (m,
5 H, aromatic); 13C NMR (acetone-d6) δ 25.42, 26.91, 30.20,
34.01, 41.34, 41.48, 122.49, 125.48, 129.83, 152.57, 155.30,
174.62.
6-[1-(4-(Methylthio)-3,5-xylyloxy)]hexanoic Acid (MXOH, Fig-
ure 2). To 50 mL of dry acetone were added stoichiometric
amounts (20 mmol) of 4-(methylthio)-3,5-xylenol, potassium
carbonate, and ethyl 6-bromohexanoate. After reflux for 12
h, the mixture was filtered and the solvent was removed under
reduced pressure. The residue was dissolved in 50 mL of ethyl
acetate, washed with water (2 × 50 mL), 1 M NaOH (2 × 50
mL), and 4 M NaCl (2 × 50 mL), and finally dried over Na2SO4.
GC analysis confirmed that 43% of the crude product was ethyl
6-[(1-(4-(methylthio)-3,5-xylyloxy)]hexanoate. After evapora-
tion of the solvent, 50 mL of 1 M NaOH was added to the
residue (4.91 g), and the solution was stirred while heated
under reflux for 1.5 h. The solution was then acidified with
concentrated hydrochloric acid, extracted with ethyl acetate,
and dried over Na2SO4. The oily product obtained after solvent
evaporation (1.55 g, 27%) was crystallized from hexane to
obtain 640 mg of the pure hapten: 1H NMR (acetone-d6) δ
1.40-1.80 (m, 6 H, CH2-CH2-CH2), 2.15 (s, 3 H, S-CH3), 2.33
(t, 2 H, CH2-COOH), 2.49 (s, 6 H, 2 CH3), 3.40 (t, 2 H, O-CH2),
6.72 (s, 2 H, aromatic); 13C NMR (acetone-d6) δ 18.60, 21.97,
25.34, 26.29, 34.06, 68.12, 114.89, 126.59, 144.82, 159.76,
174.61.
From appropriate spacers, the following compounds were
synthesized also by O-alkylation of 4-(methylthio)-3,5-xylenol.
2-[1-(4-(Methylthio)-3,5-xylyloxy)]acetic Acid (MXOA): 1H
NMR (acetone-d6) δ 2.16 (s, 3 H, S-CH3), 2.50 (s, 6 H, 2 CH3),
4.70 (s, 2 H, CH2-COOH), 6.75 (s, 2 H, aromatic); 13C NMR
(acetone-d6) δ 18.51, 21.98, 65.04, 114.93, 127.62, 144.94,
158.66, 170.07.
4-[1-(4-(Methylthio)-3,5-xylyloxy)]butanoic Acid (MXOB): 1H
NMR (acetone-d6) δ 2.04 (m, 2 H, CH2-CH2-CH2), 2.15 (s, 3
H, S-CH3), 2.49 (t, 2 H, CH2-COOH), 2.49 (s, 6 H, 2 CH3),
4.02 (t, 2 H, O-CH2), 6.74 (s, 2 H, aromatic); 13C NMR (acetone-
d6) δ 18.58, 21.97, 25.34, 67.30, 114.90, 126.79, 144.86, 159.58,
174.32.
Cell Fusion. P3-X63/Ag 8.653 murine myeloma cells (ATCC,
Rockville, MD) were cultured in high-glucose DMEM supple-
mented with 2 mM L-glutamine, 1 mM nonessential amino
acids, 25 µg/mL gentamicin, and 15% fetal bovine serum
(referred to as s-DMEM). Cell fusion procedures were carried
out essentially as described by Nowinski et al. (1979). Mouse
spleen lymphocytes were fused with myeloma cells at a 5:1
ratio using PEG 1500 as the fusing agent. The fused cells were
distributed in 96-well culture plates at an approximate density
of (4-5) × 105 cells/well in 100 µL of s-DMEM. Twenty-four
hours after plating, 100 µL of HAT selection medium (s-DMEM
supplemented with 100 µM hypoxanthine, 0.4 µM aminopter-
ine, and 16 µM thymidine) was added to each well. Half the
medium of the wells was replaced by fresh HAT medium on
day 4 postfusion and by HT medium (HAT medium without
aminopterine) on day 8 postfusion.
P r ep a r a t ion of Im m u n izin g Con ju ga t es. Haptens
MXNP, MXNB, and MXNH were covalently attached to BSA
using the modified active ester method (Langone and Van
Vunakis, 1982). Twenty-five micromoles of the hapten was
incubated overnight at room temperature with stoichiometric
amounts of N-hydroxysuccinimide and dicyclohexylcarbodi-
imide in 0.5 mL of DMF. After centrifuging, 400 µL of the
clear supernatant containing the active ester was slowly added
to 2 mL of a 15 mg/mL BSA solution in 50 mM carbonate
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 Sephadex G-50 using 100 mM
sodium phosphate buffer, pH 7.4, as eluant. Conjugate
formation was confirmed spectrophotometrically. UV-vis
spectra showed qualitative differences between the carrier
protein and conjugates in the region of maximum absorbance
of haptens. The hapten to protein molar ratio of conjugates
was then estimated from the spectral data of the hapten, the
Hybridoma Selection and Cloning. Eight to 11 days after
cell fusion, culture supernatants were screened for the pres-
ence of antibodies that recognized methiocarb. The screening
consisted of the simultaneous performance of a noncompetitive
and a competitive indirect ELISA, to test the ability of
antibodies to bind the OVA conjugate of the immunizing

Monoclonal Antibodies to Methiocarb
J. Agric. Food Chem., Vol. 46, No. 6, 1998 2421
hapten and to recognize methiocarb, respectively. For each
culture supernatant, the signal obtained in noncompetitive
conditions was compared with the competitive one, and the
ratio of both absorbances was used as the criterion for selecting
high-affinity antibody-secreting clones. Selected hybridomas
were cloned by limiting dilution on a feeder layer of BALB/c
thymocytes (∼106 cells/well) and peritoneal macrophages
(∼5000 cells/well). Stable antibody-producing clones were
expanded and cryopreserved in liquid nitrogen.
haptens resembling as much as possible the structure
and electronic distribution of methiocarb is a necessary
and critical step in the production of high-affinity
antibodies. Accordingly, we synthesized three im-
munizing haptens, namely MXNP, MXNB, and MXNH,
by the introduction of spacer arms of different lengths
through the carbamate group characteristic of this
pesticide (Figure 1). In this way, that is, by elongation
of an aliphatic chain already present in the analyte, both
the structure and the electronic distribution of the
methiocarb molecule are hardly modified, as demos-
trated by the high-affinity MAbs previously obtained
following this chemical approach for carbaryl (Abad and
Montoya, 1994), carbofuran (Abad et al., 1997c), and
bendiocarb (unpublished results).
Apart from the immunizing haptens required for
antibody production, the development of an ELISA for
pesticides relies on the synthesis of proper assay hap-
tens. These haptens can be the same used to derive the
antibodies (homologous haptens) or different ones (het-
erologous haptens). Although excellent immunoassays
may be developed using homologous haptens, especially
with MAbs, the synthesis of heterologous haptens is
usually a very valuable approach to improve the sen-
sitivity of immunoassays for pesticides. In this work
this possibility has been tested by synthesizing a panel
of 15 haptens, grouped into 5 series, that differ from
the immunizing haptens in the number of ring substit-
uents (MCN*, DPN*, MPN*, and PN* series; Figure 1)
or in the type of spacer arm (MXO* series; Figure 2).
Haptens in the same series differ from one another in
the spacer arm length. Thus, *P haptens have a spacer
arm derived from aminop ropanoic acid, *B haptens have
a spacer arm derived from aminobutyric acid, and *H
haptens have a spacer arm derived from aminoh exanoic
acid.
Purification of MAbs. Antibodies were purified directly from
late stationary phase culture supernatants by ammonium
sulfate precipitation followed by anion-exchange chromatog-
raphy on DEAE-Sepharose (Sigma). Most culture super-
natants were able to provide enough MAb (3-10 mg/100 mL)
for characterization studies and further work. Purified MAbs
were stored at 4 °C as ammonium sulfate precipitates.
En zym e-Lin ked Im m u n osor ben t Assays (ELISAs). Flat-
bottom polystyrene ELISA plates were coated overnight with
conjugate or antibody solutions in 50 mM carbonate buffer,
pH 9.6. Standards were prepared in PBS by serial dilutions
from a stock solution in DMF, using borosilicate glass tubes.
A volume of 100 µL per well was used throughout all assay
steps, and all incubations were carried out at room tempera-
ture. After each incubation, plates were washed four times
with washing solution (0.15 M NaCl containing 0.05% Tween
20). Two basic formats were used depending on the assay
component immobilized into the ELISA plates. In the conju-
gate-coated format, an indirect ELISA was used to estimate
mouse serum antibody titers and for the screening of culture
supernatants, and a competitive indirect ELISA was used for
the study of antibody sensitivity and specificity to methiocarb.
In the antibody-coated format, the specific antibody was coated
directly or by using a capture auxiliary antibody, and competi-
tive ELISAs were followed to evaluate the assay properties
using different enzyme tracers (hapten-HRP conjugates).
For competition assays, optimum concentrations of antibod-
ies, hapten conjugates, or enzyme tracers were previously
determined. Competitive curves were obtained by plotting
absorbance against the logarithm of analyte concentration.
Sigmoidal curves were fitted to a four-parameter logistic
equation (Raab, 1983), using Sigmaplot software package
(J andel Scientific, Germany).
P r od u ction of An tibod ies to Meth ioca r b. Mouse
Polyclonal Response. To evaluate the suitability of the
synthesized haptens to raise anti-methiocarb antibodies,
three mice were immunized with each of the BSA
conjugates of the MXN* hapten series. After three
injections, the titer (serum dilution giving an absorbance
of 1.0 in the established conditions) of antibodies
recognizing conjugated haptens was estimated by indi-
rect ELISA using the OVA-MXN* conjugates as coating
antigens. The three immunogens showed anti-conju-
gate reactivity with titers around 1 × 105 for the three
coating conjugates, so no influence of the spacer arm
length was observed at this point. Sera were subse-
quently tested for their ability to recognize methiocarb
by indirect competitive ELISA. All sera bound com-
petitively to methiocarb with a concentration producing
50% inhibition of antibody binding (I50) in the high
nanomolar range (Table 1). No noticeable influence of
the hapten spacer arm length was observed. Thus, the
three haptens seem to be equally suitable for the
production of MAbs to methiocarb. Accordingly, cell
fusions were performed with the nine immunized mice,
and the obtained results are summarized in Table 1.
All of the fusions rendered wells with antibodies rec-
ognizing the corresponding homologous conjugated hap-
tens, but usually only a few of these wells recognized
0.1 µM methiocarb in competitive assays. From com-
petitive wells, 12 hybridomas showing the highest
affinity to methiocarb were finally cloned and stabi-
lized: 2 hybridomas came from the hapten with the
shortest spacer arm (MXNP), 2 came from the hapten
Conjugate-Coated Format. Plates were coated with 1 µg/
mL of OVA-hapten conjugates. Different antibody concentra-
tions in PBS containing 0.1% BSA (PBSB) were then added
and incubated for 2 h. Next, plates were incubated for 1 h
with peroxidase-labeled rabbit anti-mouse immunoglobulins
diluted 1/2000 in PBST (PBS containing 0.05% Tween 20).
Finally, peroxidase activity bound to the wells was determined
by adding the substrate solution (2 mg/mL OPD and 0.012%
H2O2 in 25 mM citrate, 62 mM sodium phosphate, pH 5.4).
After 10 min, the reaction was stopped with 2.5 M sulfuric
acid, and the absorbance at 490 nm was read and recorded.
For competitive assays, the procedure was the same except
that after coating, a competition step was introduced by adding
50 µL of methiocarb standards followed by 50 µL of the
appropriate concentration of antibody.
Antibody-Coated Format. In this format, plates were coated
with antibodies at 1 µg/mL. Next, the competition was
established for 1 h between methiocarb standards and the
selected concentrations of enzyme tracers. Peroxidase activity
was measured as above.
Indirect Antibody-Coated Format. The difference from the
previous format was that plates were first coated with goat
anti-mouse immunoglobulins at 2 µg/mL in carbonate buffer,
followed by an incubation for 2 h with the specific antibodies
at 1 µg/mL in PBST.
RESULTS AND DISCUSSION
Syn th esis of Ha p ten s. Methiocarb, like most pes-
ticides, is a small and simple organic molecule non-
immunogenic by itself and lacking a functional group
for coupling to proteins. Therefore, the synthesis of

2422 J. Agric. Food Chem., Vol. 46, No. 6, 1998
Abad et al.
Ta ble 1. Su m m a r y of Cell F u sion a n d Hybr id om a Selection Resu lts
no. of wells
immunizing
hapten
seruma
I50 (nM)
positiveb
(conjugate)
competitivec
(analyte)
no. of cloned
fusion no.
seeded
hybridomasd
MXNP
MXNB
MXNH
1
2
3
1
2
3
1
2
3
329
121
428
618
156
1128
273
713
694
288
288
288
288
288
384
384
288
288
22
46
150
14
2
46
0
1
0
16
12
20
0
2e
2e
0
1
0
2
4
4
0
4
177
150
141
4
a
ELISA plates were coated with 1 µg/mL of the homologous conjugate, and the antiserum dilution used was previously determined to
b
obtain a maximum absorbance around 1.0. Wells with antibodies that recognized the OVA-hapten conjugates (homologous assays) by
indirect ELISA (absorbance > 0.5). c Wells with antibodies that recognized 0.1 µM methiocarb in solution (inhibition > 50%). Only
d
hybridomas secreting antibodies with the lowest I50 for methiocarb were stabilized and cloned. e Indicates that these antibodies were
rejected during the cloning proccess because better antibodies were obtained in further fusions.
Ta ble 2. Recogn ition of Ha p ten s Cou p led to Ova lbu m in by An ti-Meth ioca r b MAbs in th e Con ju ga te-Coa ted F or m a ta
MAb
conjugated
hapten
LIB-MXNP22
LIB-MXNB31
LIB-MXNB33
LIB-MXNH14
LIB-MXNH15
LIB-MXNH22
MXNP
MXNB
MXNH
MCNP
MCNB
MCNH
DPNP
DPNB
DPNH
MPNP
MPNB
MPNH
PNP
+
+
+
+
+
+
+
+
+
+
+
+
(
+
+
+
+
+
(
+
+
(
+
+
+
+
+
(
+
+
+
+
+
(
(
+
+
(
(
+
+
+
(
(
PNB
PNH
MXOA
MXOB
MXOH
+
+
+
+
a
ELISA plates were coated with 1 µg/mL of OVA conjugates of haptens displayed in the table. Symbols indicate the antibody
concentration that produces absorbances of 1.0 in the established conditions: <0.02 µg/mL (+); between 0.02 and 0.3 µg/mL ((); >0.3
µg/mL (no entry).
with the medium-sized spacer arm (MXNB), and 8 came
from the hapten with the largest spacer arm (MXNH).
The fact that the highest number of hybridomas was
derived from the MXNH hapten should not be neces-
sarily attributed to the spacer arm length but rather to
the high intrinsic variability of the cell fusion process.
MAbs obtained were small-scale-purified from culture
supernatant, and their ability to recognize methiocarb
was roughly estimated from inhibition curves in the
conjugate-coated ELISA format using homologous hap-
tens. Under these conditions, I50 values in the 1-10
nM range were obtained, which means an improvement
of 2 orders of magnitude with respect to the affinity
displayed by mouse antisera evaluated in the same
conditions. According to these data, a group of six MAbs
displaying the lowest I50 values were selected for further
studies using the complete panel of synthesized haptens
coupled either to OVA (conjugate-coated format) or to
HRP (antibody-coated format and indirect antibody-
coated format).
obtain the MAbs. The obtained results are summarized
in Table 2, and they essentially show a very different
recognition pattern of OVA-hapten conjugates by each
MAb. Thus, whereas LIB-MXNP22 MAb recognized
only 3 immobilized conjugates, LIB-MXNB31 MAb
recognized 12 of 18 conjugates. As would be expected,
the best recognized conjugates by all MAbs were those
derived from haptens belonging to the MXN* series,
since they are homologous or differ only in the spacer
arm length. On the opposite side, conjugates derived
from haptens of the PN* series, which lack all ring
substituents, were not recognized by any of the MAbs,
and conjugates from haptens with only one ring sub-
stituent (MPN* series) were well recognized only by
LIB-MXNB31 MAb, the antibody less sensitive to
changes in the assay hapten structure. An intermediate
situation was found for immobilized conjugates from
haptens with two ring substituents (MCN* and DPN*
series), because they were recognized with a high titer
by most antibodies, especially those haptens with spacer
arms of 4-6 carbon atoms. On the other hand, haptens
of the MXO* series, with no modifications in the
aromatic structure but with the spacer arm directly
attached to the oxygen atom of the phenolic precursor,
were recognized only by LIB-MXNB31 MAb. This
finding calls into question the general usefulness of this
promising chemical approach to obtain heterologous
Ch a r a cter iza tion of th e MAbs in th e Con ju ga te-
Coa ted F or m a t. Recognition of Conjugated Haptens.
Before inhibition experiments were performed, the six
selected MAbs were evaluated for their ability to
recognize conjugates of ovalbumin with haptens dis-
playing modifications in the aromatic moiety and/or in
the spacer arm, with respect to the compounds used to

Monoclonal Antibodies to Methiocarb
J. Agric. Food Chem., Vol. 46, No. 6, 1998 2423
Ta ble 3. Stu d y of th e In flu en ce of ELISA F or m a t a n d Ha p ten Heter ology w ith An ti-Meth ioca r b MAbsa
LIB-MXNB31
LIB-MXNB33
LIB-MXNH14
LIB-MXNH15
format
AC
format
AC
format
AC
format
AC
conjugated
hapten
CC
IAC
CC
IAC
CC
IAC
CC
IAC
MXNB
MXNH
MCNH
DPNH
I50
Amax
I50
Amax
I50
Amax
I50
1.65
0.86
1.13
1.04
0.21
1.41
0.12
1.13
0.53
1.44
0.92
1.90
0.60
2.26
0.33
0.93
0.92
1.13
0.93
1.26
0.22
0.83
0.62
0.87
0.89
1.02
0.39
0.85
0.80
1.10
1.43
1.19
0.69
1.14
0.53
0.84
0.85
1.22
0.91
1.32
0.14
1.44
0.22
1.22
0.47
0.84
0.63
0.95
0.47
1.21
0.32
1.19
0.46
1.34
0.69
1.39
0.14
1.15
0.14
0.97
0.39
0.48
0.11
0.76
0.61
0.68
0.30
1.03
0.23
0.81
0.81
0.87
0.53
1.14
0.31
1.01
0.20
0.76
0.19
0.90
Amax
a
I50 values are expressed in nanomolar, Amax was measured at 490 nm, and homologous combinations are indicated in boldface print.
No entry indicates that this particular combination was not tested because it did not provide a sufficient maximum absorbance in the
b
absence of the analyte. CC, conjugate-coated format; AC, antibody-coated format; IAC, indirect antibody-coated format.
haptens for this class of pesticides and emphasizes the
importance of maintaining the carbamate group in the
structure of the assay hapten.
As a conclusion, although the most potentially suit-
able assay haptens were those involving slight modifica-
tions of the immunizing hapten structure, the relation-
ship between the hapten structure and its uselfulness
as an assay hapten is strongly dependent on the
antibody. Therefore, the availability of a wide panel of
antibodies is essential to study such a basic question in
immunoassay development.
Affinity. Heterologous assays are well-known proce-
dures to improve the sensitivity of immunoassays,
because the structure of the assay hapten modulates
F igu r e 3. Standard curves for methiocarb obtained with the
MAb LIB-MXNB31 (0.015 µg/mL) in the conjugate-coated
format, using the assay haptens MXNB (b) and DPNH (9)
coupled to ovalbumin at 1 µg/mL. Experimental points,
obtained in quadruplicate, were fitted to a four-parameter
logistic equation. Enzymatic reactions were stopped after 10
min.
the equilibrium conditions of the competition (Harrison
et al., 1991b; Marco et al., 1995). However, it should
be also taken into account that it is nearly impossible
to predict the structure of the hapten that will provide
a more sensitive assay, as well as the extent of the
improvement achievable (Abad et al., 1997a). There-
fore, after the number of conjugates and the extent at
which they were recognized by each MAb were deter-
mined, inhibition curves using methiocarb as competitor
were performed for all coating conjugates that were
recognized by each antibody.
Most MAbs increased their apparent affinity to me-
thiocarb by using heterologous conjugates, with im-
provements in sensitivity (I50 values) of up to 1 order of
magnitude (Table 3, CC format). Thus, the I50 value of
the MAb LIB-MXNB31 changed from 1.65 nM with the
OVA-MXNB conjugate (homologous hapten) to 0.12 nM
with the OVA-DPNH conjugate (Figure 3). Sensitivity
improvements of 4-6 times were also observed with
antibodies LIB-MXNB33, -MXNH14, and -MXNH15
(Table 3). However, no noticeable improvement was
found with LIB-MXNP22 and -MXNH22 MAbs (data not
shown). A general finding was that assay haptens of
the same series, that is, haptens differing only in the
spacer arm length, provide immunoassays with very
similar I50 values. Therefore, although haptens with
spacer arm length heterology are normally easily pre-
pared, great sensitivity improvements should not be
expected by following this strategy.
values were used to calculate cross-reactivities. 4-(Meth-
ylthio)-3,5-xylenol lacks the methylcarbamate group
characteristic of methiocarb, and methiocarb sulfoxide
and sulfone differ from the parent insecticide only in
the oxidation state of the sulfur atom (Figure 4).
Despite the great structural similarity between methio-
carb and its metabolites, all of the MAbs were extra-
ordinarily specific to methiocarb. Hence, cross-reactiv-
ity values for methiocarb sulfone, the best recognized
metabolite, were 0.03% with the antibodies LIB-
MXNB31 and LIB-MXNB33 and 0.4% with the rest of
the MAbs.
Eva lu a tion of th e An tibod y-Coa ted F or m a ts. As
in the conjugate-coated format, noncompetitive experi-
ments were carried out prior to inhibition assays in the
antibody-coated formats. Concentrations of HRP-hap-
ten conjugates (enzyme tracers) from 1 ng/mL to 1 µg/
mL were tested in plates previously coated with the
MAbs at a fixed concentration of 1 µg/mL, either directly
or through a capture immunoglobulin. Most of the
HRP-hapten conjugates were not recognized by the
MAbs directly immobilized to the plate. Thus, only
enzyme conjugates derived from the haptens MXNB,
MXNH, MCNH, and DPNH were recognized, and not
even by all of the MAbs. In fact, LIB-MXNP22 and
-MXNH22 MAbs did not bind any of the 12 HRP-
hapten conjugates assayed, LIB-MXNB31 and
-MXNH14 MAbs recognized 2 conjugates, and LIB-
MXNH15 and -MXNB33 MAbs recognized 3 conjugates.
It should be noticed that the MAb that recognized more
coating conjugates (LIB-MXNB31) is not the antibody
Specificity. The specificity of the MAbs was evaluated
in the conjugate-coated format using their respective
homologous hapten, that is, OVA-MXNP for LIB-
MXNP22 MAb, OVA-MXNB for LIB-MXNB31 and
-MXNB33 MAbs, and OVA-MXNH for LIB-MXNH14,
-MXNH15, and -MXNH22 MAbs. Competitive assays
with the methiocarb metabolites 4-(methylthio)-3,5-
xylenol, methiocarb sulfoxide, and methiocarb sulfone
as competitors were performed, and the obtained I50

2424 J. Agric. Food Chem., Vol. 46, No. 6, 1998
Abad et al.
F igu r e 5. Standard curves for methiocarb using enzyme
conjugates of the haptens MXNH (b, 0.3 µg/mL) and DPNH
(9, 0.5 µg/mL) obtained with the MAb LIB-MXNH15 (1 µg/
mL) in the antibody-coated format. Experimental points,
obtained in quadruplicate, were fitted to a four-parameter
logistic equation. Enzymatic reactions were stopped after 10
min.
F igu r e 4. Chemical structures of methiocarb and methiocarb
metabolites.
that bound more enzyme tracers and that LIB-MXNH14
and -MXNB31 MAbs recognized only heterologous en-
zyme conjugates. These results agree with previous
authors’ experience in this field, which indicates that
the number of haptens coupled to HRP recognized by
immobilized antibodies is always lower than the number
of haptens recognized when coupled to OVA in the
conjugate-coated format by the same antibodies (Man-
clu´s and Montoya, 1996; Manclu´s et al., 1996; Abad et
al., 1997a,b). Moreover, it is very infrequent that an
immobilized MAb recognizes a hapten conjugated to
HRP if this hapten, coupled to OVA, is not recognized
by the same antibody in the conjugate-coated format.
The fact that none of the haptens with spacer arms
derived from aminopropanoic acid (*P haptens), and
only one of those containing aminobutyric acid (MXNB
hapten), was recognized by the immobilized antibodies
agree with previous results reported by other authors
indicating that haptens with short linking groups are
less suitable for enzyme conjugates, probably due to
steric hindrances (Skerrit et al., 1992; Bekheit et al.,
1993; Schneider et al., 1994).
Methiocarb inhibition curves were performed for all
enzyme conjugate/MAb combinations that provided a
sufficient signal. I50 values in the 0.1-0.9 nM range
were obtained (Table 3, AC format), which were very
similar to those found in the conjugate-coated format
(Table 3, CC format). Concerning hapten heterology,
assay sensitivity in the antibody-coated format also
improved by the use of heterologous enzyme conjugates
with respect to that achieved with homologous enzyme
conjugates, although at a lower extent than in the
conjugate-coated format. Thus, a 3-fold I50 decrease was
observed with LIB-MXNH15 MAb (Figure 5).
F igu r e 6. Methiocarb competitive curves obtained for LIB-
MXNH15 MAb in combination with the heterologous assay
hapten MCNH in the three formats tested: conjugate-coated
format (b), antibody-coated format (9), and indirect antibody-
coated format (2). Experimental points, obtained in quadru-
plicate, were fitted to a four-parameter logistic equation.
Enzymatic reactions were stopped after 10 min.
four enzyme conjugates but also improved their affinity
to the enzyme tracers previously recognized when
directly immobilized to the plate. With respect to assay
sensitivity, I50 values were in most cases very similar
to those found in the other two assay formats (Table 3).
In this respect, it is interesting to point out that only
slight differences among assay configurations were
observed, as perfectly exemplified by the MAb LIB-
MXNH15 in combination with the assay hapten MCNH
(Figure 6).
CONCLUSIONS
The goal of obtaining a wide panel of high-affinity
MAbs to the N-methylcarbamate pesticide methiocarb
was achieved from mice immunized with BSA conju-
gates of three haptens derivatized at the carbamate
group and differing in the spacer arm length. In the
conjugate-coated format with homologous coating con-
jugates, MAbs exhibited I50 values in the 1-10 nM
range. This result, along with previous experience with
other carbamates, confirms the excellence of this hapten
synthesis strategy as a general and simple approach for
the obtention of antibodies to this family of pesticides.
Competitive assays with methiocarb metabolites evi-
The effect of a precoating step with goat anti-mouse
Igs (indirect antibody-coated format) was also evaluated
as a well-known procedure to improve the ability of
immobilized antibodies to recognize enzyme conjugates
(Schneider and Hammock, 1992; Giersch, 1993; Manclu´s
et al., 1996; Abad et al., 1997a,b). This approach proved
to be especially successfull for the MAbs LIB-MXNB31
and -MXNH14, which not only recognized in this way

Monoclonal Antibodies to Methiocarb
J. Agric. Food Chem., Vol. 46, No. 6, 1998 2425
Abad, A.; Primo, J .; Montoya, A. Development of an enzyme-
linked immunosorbent assay to carbaryl. 1. Antibody pro-
duction from several haptens and characterization in dif-
ferent immunoassay formats. J . Agric. Food Chem. 1997a ,
45, 1486-1494.
Abad, A.; Manclu´s, J . J .; Mojarrad, F.; Mercader, J . V.;
Miranda, M. A.; Primo, J .; Guardiola, V.; Montoya, A.
Hapten synthesis and production of monoclonal antibodies
to DDT and related compounds. J . Agric. Food Chem. 1997b,
45, 3694-3702.
Abad, A.; Moreno, M. J .; Montoya, A. A monoclonal immuno-
assay for carbofuran and its application to the analysis of
fruit juices. Anal. Chim. Acta 1997c, 347, 103-110.
Bakowski, R. S.; Ali, M. S.; White, J . D.; Phillippo, E. T.; Ellis,
R. L. Gas chromatographic/mass spectrometric confirmation
of ten N-methylcarbamate insecticides in liver. J . AOAC Int.
1994, 77, 1568-1574.
Ballesteros, E.; Gallego, M.; Valca´rcel, M. Automatic gas
chromatographic determination of N-methylcarbamates in
milk with electron capture detection. Anal. Chem. 1993, 65,
1773-1778.
Bekheit, H. K. M.; Lucas, A. D.; Szurdoki, F.; Gee, S. J .;
Hammock, B. D. An enzyme immunoassay for the environ-
mental monitoring of the herbicide bromacil. J . Agric. Food
Chem. 1993, 41, 2220-2227.
Chiron, S.; Torres, J . A.; Ferna´ndez-Alba, A.; Alpendurada,
M. F.; Barcelo´, D. Identification of carbofuran and methio-
carb and their transformation products in estuarine waters
by on-line solid-phase extraction liquid chromatography-
mass spectrometry. Int. J . Environ. Anal. Chem. 1996, 65,
37-52.
Dankwardt, A.; Hock, B. Enzyme immunoassays for analysis
of pesticides in water and food. Food Technol. Biotechnol.
1997, 35, 164-174.
Di Corcia, A.; Crescenzi, C.; Lagana`, A. Evaluation of a method
based on liquid chromatography/electrospray/mass spec-
trometry for analyzing carbamate insecticides in fruits and
vegetables. J . Agric. Food Chem. 1996, 44, 1930-1938.
Dolbeer, R. A.; Avery, M. L.; Tobin, M. E. Assessment of field
hazards to birds from methiocarb applications to fruit crops.
Pestic. Sci. 1994, 40, 147-161.
Fa¨rber, H.; Scho¨ler, H. F. Gas chromatographic determination
of carbamate pesticides after flash-heater methylation with
trimethylsulfonium hydroxide. J . Agric. Food Chem. 1993,
41, 217-220.
Giersch, T. A new monoclonal antibody for the sensitive
detection of atrazine with immunoassay in microtiter plate
and dipstick format. J . Agric. Food Chem. 1993, 41, 1006-
1011.
Goodrow, M. H.; Sanborn, J . R.; Stoutamire, D. W.; Gee, S. J .;
Hammock, B. D. Strategies for immunoassay hapten design.
In Immunoassays for Agrochemicals. Emerging Technolo-
gies; Nelson, J . O., Karu, A. E., Wong, R. B., Eds.; ACS
Symposium Series 586; American Chemical Society: Wash-
ington, DC, 1995; pp 140-152.
Gunderson, E. L. FDA total diet study, J uly 1986-April 1991,
dietary intakes of pesticides, selected elements, and other
chemicals. J . AOAC Int. 1995, 78, 1353-1363.
Hammock, B. D.; Gee, S. J . Impact of emerging technologies
on immunochemical methods for environmental analysis.
In Immunoassays for Agrochemicals. Emerging Technolo-
gies; Nelson, J . O., Karu, A. E., Wong, R. B., Eds.; ACS
Symposium Series 586; American Chemical Society: Wash-
ington, DC, 1995; pp 1-19.
Harrison, R. O.; Goodrow, M. H.; Gee, S. J .; Hammock, B. D.
Hapten synthesis for pesticide immunoassay development.
In Immunoassays for Trace Chemical Analysis; Vanderlaan,
M., Stanker, L. K., Watkins, B. E., Roberts, D. W., Eds.;
ACS Symposium Series 451; American Chemical Society:
Washington, DC, 1991a; pp 14-27.
denced the great specificity to methiocarb of most of the
MAbs obtained. Thus, the absence of the methyl-
carbamate group or the oxidation of the sulfur atom
severely decreases the affinity of the antibodies to these
compounds.
With the idea in mind of a further improvement in
assay sensitivity, 15 heterologous haptens with modi-
fications in the aromatic moiety or in the spacer arm
were synthesized and evaluated, and the influence of
the ELISA format on the assay sensitivity was studied.
Our results completely confirm that hapten heterology
is a valuable approach to improve the sensitivity of
immunoassays for pesticides, as evidenced by decreases
in I50 values of up to 1 order of magnitude. The best
heterologous haptens were those with minor modifica-
tions in the ring structure and with long spacer arms
of the same type of the immunizing hapten, particularly
in the antibody-coated formats. Nevertheless, haptens
with major modifications were perfectly adequate for
certain antibodies. Very similar sensitivities were
obtained in the different assay formats. The main
difference among ELISA formats is the number of
conjugated haptens suitable for the development of
immunoassays, since more haptens were recognized by
the antibodies when coupled to OVA and used in the
conjugate-coated format than when coupled to HRP and
used in the antibody-coated formats.
To our knowledge, the MAbs for methiocarb herein
presented are the first reported for this pesticide. The
high affinity to methiocarb displayed by some combina-
tions of antibody, assay hapten, and ELISA format, with
I50 values in the 0.1-0.3 nM range, is one of the highest
ever reported for an antipesticide antibody, making
them very promising immunoreagents for the develop-
ment of a sensitive and specific immunoassay for this
agrochemical contaminant. In fact, with regard to the
sensitivity reached by these immunoreagents, it seems
highly feasible to develop an immunoassay able to
directly determine residues of methiocarb as low as 5
ppb in fruit and vegetable juices, without sample
concentration and/or cleanup and with the only need of
a 100-fold sample dilution to enter into the working
range of the ELISA standard curve. Therefore, work
is in progress to select the most sensitive of the
immunoreagent combinations for each assay format and
to assess the influence of several physicochemical factors
on their performance, as well as to study their suit-
ability for the analysis of methiocarb residues in agri-
cultural and environmental samples.
ABBREVIATIONS USED
BSA, bovine serum albumin; CR, cross-reactivity;
DMEM, Dulbecco’s Modified Eagle’s Medium; DMF,
N,N-dimethylformamide; ELISA, enzyme-linked immu-
nosorbent assay; HRP, horseradish peroxidase; I50
,
concentration giving 50% inhibition of maximum re-
sponse; Ig, immunoglobulin; MAb, monoclonal antibody;
NMR, nuclear magnetic resonance; OPD, o-phenylene-
diamine; OVA, ovalbumin; PBS, phosphate-buffered
saline; PBSB, phosphate-buffered saline containing
0.1% BSA; PBST, phosphate-buffered saline containing
0.05% Tween 20; PEG, poly(ethylene glycol); UV-vis,
ultraviolet-visible.
LITERATURE CITED
Harrison, R. O.; Goodrow, M. H.; Hammock, B. D. Competitive
Inhibition ELISA for the S-Triazine Herbicides-Assay Op-
timization and Antibody Characterization. J . Agric. Food
Chem. 1991b, 39, 122-128.
Abad, A.; Montoya, A. Production of monoclonal antibodies for
carbaryl from a hapten preserving the carbamate group. J .
Agric. Food Chem. 1994, 42, 1818-1823.

2426 J. Agric. Food Chem., Vol. 46, No. 6, 1998
Abad et al.
Krause, R. T. Liquid chromatographic determination of N-
methylcarbamate insecticides and metabolites in crops. I.
Collaborative study. J . Assoc. Off. Anal. Chem. 1985, 68,
726-733.
Langone, J . J .; Van Vunakis, H. Radioimmunoassay of nico-
tine, cotinine, and γ-(3-piridyl)-γ-oxo-N-methylbutyramide.
Methods Enzymol. 1982, 84, 628-640.
Raab, G. M. Comparison of a logistic and a mass-action curve
for radioimmunoassay data. Clin. Chem. 1983, 29, 1757-
1761.
Rajkowski, K. M.; Cittanova, N.; Desfosses, B.; J ayle, M. F.
The conjugation of testosterone with horseradish peroxidase
and a sensitive enzyme assay for the conjugate. Steroids
1977, 29, 701-713.
Lisˇka, I.; Slobodn´ık, J . Comparison of gas and liquid chroma-
tography for analysing polar pesticides in water samples.
J . Chromatogr. A 1996, 733, 235-258.
Manclu´s, J . J .; Montoya, A. Development of an enzyme-linked
immunosorbent assay for 3,5,6-trichloro-2-pyridinol. 1. Pro-
duction and characterization of monoclonal antibodies. J .
Agric. Food Chem. 1996, 44, 3703-3709.
Manclu´s, J . J .; Primo, J .; Montoya, A. Development of an
enzyme-linked immunosorbent assay for the insecticide
chlorpyrifos. 1. Monoclonal antibody production and immu-
noassay design. J . Agric. Food Chem. 1996, 44, 4052-4062.
Marco, M. P.; Gee, S. J .; Hammock, B. D. Immunochemical
techniques for environmental analysis II. Antibody produc-
tion and immunoassay development. Trends Anal. Chem.
1995, 14, 415-425.
McGarvey, B. D. High-performance liquid chromatographic
methods for the determination of N-methylcarbamate pes-
ticides in water, soil, plants and air. J . Chromatogr. 1993,
642, 89-105.
Meulenberg, E. P.; Mulder, W. H.; Stoks, P. G. Immunoassays
for pesticides. Environ. Sci. Technol. 1995, 29, 553-561.
Miller, F. K.; Kiigemagi, U.; Thomson, P. A.; Heatherbell, D.
A.; Deinzer, M. L. Methiocarb residues in grapes and wine
and their fate during vinification. J . Agric. Food Chem.
1985, 33, 538-545.
Moye, H. A.; Scherer, S. J .; St. J ohn, P. A. Dynamic fluorogenic
labeling of pesticides for high performance liquid
chromatography: detection of N-methylcarbamates with
o-phthaldehyde. Anal. Lett. 1977, 10, 1049-1073.
Mueller, H. M.; Stan, H. J . Thermal degradation observed with
different injection techniques: quantitative estimation by
the use of thermolabile carbamate pesticides. J . High
Resolut. Chromatogr. 1990, 13, 759-763.
Schneider, P.; Hammock, B. D. Influence of the ELISA format
and the hapten enzyme conjugate on the sensitivity of an
immunoassay for s-triazine herbicides using monoclonal
antibodies. J . Agric. Food Chem. 1992, 40, 525-530.
Schneider, P.; Goodrow, M. H.; Gee, S. J .; Hammock, B. D. A
Highly sensitive and rapid ELISA for the arylurea herbi-
cides diuron, monuron, and linuron. J . Agric. Food Chem.
1994, 42, 413-422.
Skerritt, J . H.; Hill, A. S.; Beasley, H. L.; Edward, S. L.;
Mcadam, D. P. Enzyme-linked immunosorbent assay for
quantitation of organophosphate pesticidessfenitrothion,
chlorpyrifos-methyl, and pirimiphos-methyl in wheat grain
and flour-milling fractions. J . AOAC Int. 1992, 75, 519-
528.
Slovodnik, J .; J ager, M. E.; Hoekstra-Oussoren, J . F.; Honing,
M.; van Baar, B. L. M.; Brinkman, U. A. Th. Identification
of carbamates by particle beam/mass spectrometry. J . Mass
Spectrom. 1997, 32, 43-54.
Szurdoki, F.; Bekheit, H. K. M.; Marco, M. P.; Goodrow, M.
H.; Hammock, B. D. Important factors in hapten design and
enzyme-linked immunosorbent assay development. In New
Frontiers in Agrochemical Immunoassay; Kurtz, D. A.,
Skerritt, J . H., Stanker, L., Eds.; AOAC International:
Arlington, VA, 1995; pp 39-63.
Worthing, C. R.; Hance, R. J . In The Pesticide Manual: A
World Compendium, 9th ed.; BCPC Publications: Berks,
U.K., 1991.
Yang, S. S.; Goldsmith, A. I.; Smetena, I. Recent advances in
the residue analysis of N-methylcarbamate pesticides. J .
Chromatogr. A 1996, 754, 3-16.
Received for review J anuary 26, 1998. Revised manuscript
received March 31, 1998. Accepted April 1, 1998. This work
was supported by a grant (ALI96-1232) from the Comisio´n
Interministerial de Ciencia y Tecnolog´ıa (Spain).
Nowinski, R. C.; Lostrom, M. E.; Tam, M. R.; Stone, M. R.;
Burnette, W. N. The Isolation of hybrid cell lynes producing
monoclonal antibodies against the p15(E) protein of ecotro-
pic murine leukemia viruses. Virology 1979, 93, 111-126.
J F980077V