Hapten Synthesis for a Monoclonal Antibody Based ELISA for Deltamethrin

Article abstract of DOI:10.1021/jf9703922

An enzyme-linked immunosorbent assay (ELISA) was developed for the insecticide deltamethrin. Two haptens were synthesized: the first one was a derivative of the whole molecule with a spacer arm bound to the aromatic ring; the second one was a derivative of deltamethric acid. Twelve monoclonal antibodies were obtained. A competitive ELISA using monoclonal antibody Del 01 yielded the most sensitive assay. The IC₅₀ value for deltamethrin was estimated to be 0.5 μg mL^-1 with a detection limit of 0.08,μg mL^-1. Monoclonal antibody Del 01 seems to be deltamethrin specific since <1% cross-reactivity with other pyrethroid analogues was observed.

Full text of DOI:10.1021/jf9703922

1670
J. Agric. Food Chem. 1998, 46, 1670−1676
Ha p ten Syn th esis for a Mon oclon a l An tibod y Ba sed ELISA for
Delta m eth r in
Anne-Laurence Queffelec,†,‡ Patrice Nodet,*,† J ean-Pierre Haelters,‡ Daniel Thouvenot,† and
Bernard Corbel‡
Laboratoire de Microbiologie et Se´curite´ Alimentaire, ESMISAB, Technopoˆle Brest-Iroise,
29280 Plouzane´, France, and Laboratoire de Chimie He´te´ro-Organique, UFR Sciences et
Techniques de Brest, B.P. 809, 29285 Brest Cedex, France
An enzyme-linked immunosorbent assay (ELISA) was developed for the insecticide deltamethrin.
Two haptens were synthesized: the first one was a derivative of the whole molecule with a spacer
arm bound to the aromatic ring; the second one was a derivative of deltamethric acid. Twelve
monoclonal antibodies were obtained. A competitive ELISA using monoclonal antibody Del 01
yielded the most sensitive assay. The IC50 value for deltamethrin was estimated to be 0.5 µg mL-1
with a detection limit of 0.08 µg mL-1. Monoclonal antibody Del 01 seems to be deltamethrin specific
since <1% cross-reactivity with other pyrethroid analogues was observed.
Keyw or d s: Deltamethrin; ELISA; hapten; monoclonal antibodies
Ta ble 1. Str u ctu r e of Sever a l P yr eth r oid s
INTRODUCTION
Deltamethrin, 1(R)-cis-R(S)-3-(2,2-dibromoethenyl)-
2,2-dimethylcyclopropane carboxylic acid cyano(3-phen-
oxyphenyl)methyl ester, is one of the most potent
insecticides known. It is a synthetic pyrethroid devel-
oped by Elliot et al. (1974). Deltamethrin is widely used
because of its excellent insecticidal properties (Elliot,
1976) and low mammalian toxicity (Mestres and Mestres,
1992). Classical methods for pyrethroid analysis (Pa-
padopoulou-Mourkidou, 1983) include gas chromatog-
raphy (Sharp et al., 1988) with electron capture detec-
tion for halogenated derivatives (Okadu et al., 1983) or
gas chromatography/mass spectrometry (Mestres et al.,
1979). These procedures are time-consuming and re-
quire expensive equipment.
To respond to the increasing number of analyzed
samples, it appeared necessary to develop fast and
economic techniques. Since Hammock and Mumma
(1980) pointed out that immunoassays could be used to
detect pesticide residues, it has been demonstrated that
immunoassays enable quick screening of samples (Hall
et al., 1990; J ung et al., 1989; Newsome, 1986).
Several mono/polyclonal-based immunoassays dealing
with pyrethroids have been reported; they included
allethrin (Pullen and Hock, 1995a,b), S-bioallethrin
(Wing et al., 1978), cypermethrin (Wraith et al., 1986),
deltamethrin (Demoute et al., 1986), bioresmethrin (Hill
et al., 1993), permethrin/1(R)-phenothrin (Stanker et al.,
1989; Skerritt et al., 1992), and permethrin (Bonwick
et al., 1994). Because of its low molecular weight,
deltamethrin, like most of pesticides, is not immuno-
genic. It must be conjugated to a carrier protein to
induce an immune response. To be bound to the carrier,
the pesticide must contain a functional group. Since
name
R1
Br
CH3
Cl
Cl
CF3
R2
Br
CH3
Cl
Cl
Cl
R3
deltamethrin
phenotrin
permethrin
cypermethrin
λ-cyalothrin
CN
H
H
CN
CN
deltamethrin has none, it was necessary to synthesize
an analogue that mimicked deltamethrin. Since an
antibody’s specificity depends on both the hapten na-
ture, that is, the entire molecule or part of it, and the
position of the binding site on the carrier protein,
different approaches could be considered.
The first one is the use of the whole molecule.
Pyrethroids (Table 1) exhibit the same chemical struc-
ture, that is, a phenoxybenzyl moiety esterified by a
molecule of chrysanthemic acid of which the ethylenic
chain is generally substituted by a halogen (Cl, Br, F).
Pyrethroid structure suggests at least two possible
positions for the binding of the spacer arm as illustrated
for deltamethrin in Table 1 (position I or II).
(i) Spacer arm binding on position I, that is, on the
ethylenic chain, leaves the aromatic ring free. Stanker
et al. (1989) used this method to obtain antibodies
against permethrin. They synthesized the required
hapten by ozonolysis of phenothrin. Monoclonal anti-
bodies produced by Stanker et al. (1989) were specific
to phenothrin and permethrin and presented cross-
reactivities of 0.5-10% with pyrethroids carrying a
cyano group. To produce antibodies against biores-
methrin, that is, a pyrethroid with a benzylfuran moiety
instead of phenoxybenzyl, Hill et al. (1993) used position
* Author to whom correspondence should be addressed
(telephone 33.2.98.05.61.24; fax 33.2.98.05.61.01; e-mail
patrice.nodet@univ-brest.fr).
† ESMISAB.
‡ UFR Sciences et Techniques de Brest.
S0021-8561(97)00392-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/05/1998

ELISA for Deltamethrin
J. Agric. Food Chem., Vol. 46, No. 4, 1998 1671
F igu r e 2. Hapten 2 synthesis pathway.
instrument. Preparative HPLC separations were performed
with an HPLC system (Spectra SP 8 800) equipped with a UV
detector (Spectra 1 000) and a preparative column containing
silica (10 µm, 10 × 250). The flow rate was 5 mL min-1
.
3-(4-Hydroxy-1-phenyl)propanoic Acid Methyl Ester (1).
Fourteen grams (77.69 mmol) of 3-(4-methoxy-1-phenyl)-
propanoic acid was stirred with 75 mL of bromohydric acid
48%. After hydrolysis, the reaction mixture was extracted
with dichloromethane. The organic layer was dried over
MgSO4 and concentrated. The residue was recrystallized from
petroleum ether and esterified by methanol in the presence
of sulfuric acid to give the methyl ester (1) (5.6 g; 40% yield):
1H NMR (CDCl3, 100.3 MHz) δ 2.57 (t, 2H, ArCH2), 2.84 (t,
2H, CH2CO2), 3.65 (s, 3H, OCH3), 6.75-7.00 (m, 4H, ArH), 7.2
(s, 1H, OH).
F igu r e 1. Hapten 1 synthesis pathway.
I to bind the spacer arm. Polyclonal antibodies obtained
were specific to the 1R trans isomer (bioresmethrin) and
presented a cross-reactivity with the 1R,S cis,trans
isomer (resmethrin).
3-[4-{3-(1,3-Dioxolan-2-yl-phenoxy)-1-phenyl}]propanoic Acid
Methyl Ester (2). A mixture composed of 4.64 g (25,75 mmol)
of compound 1, 10 mL of pyridine, and 1.4 g (25.91 mmol) of
sodium methylate was mixed and warmed to evaporate 2 mL
of pyridine, after which 0.64 g (6.46 mmol) of cuprous chloride
and 9.80 g (42.78 mmol) of 2-(3-bromophenyl)-1,3-dioxolane
were added. The mixture was vigorously stirred and warmed
to 200 °C (external temperature), while pyridine was elimi-
nated. This temperature was maintained for 8 h. The residue
was dissolved with ether and washed with an aqueous solution
of 1 M HCl and then with water. The organic layer was dried
over MgSO4 and concentrated. The residue was purified on a
silica gel column using ethyl acetate/hexane (1:6) as eluent.
The solution was washed with 1 M NaOH and extracted twice
with ether to give compound 2 (3.91 g; 46% yield): 1H NMR
(CDCl3, 100.3 MHz) δ 2.6 (t, 2H, CH2Ar), 2.9 (t, 2H, CH2CO),
3.62 (s, 3H, CH3O), 4.00 (m, 4H, CH2 dioxolane), 5.7 (s, 1H,
CH dioxolane), 6.7-7.5 (m, 8H, ArH).
3-[4-{3-(1,3-Dioxolan-2-yl-phenoxy)-1-phenyl}]propanoic Acid
(3). Fifteen milliliters of an aqueous solution of potassium
hydroxide (0.78 g, 13.90 mmol) was added to a solution of 2
(3.84 g, 11.70 mmol) in 30 mL of methanol. The reaction
mixture was stirred for 20 h at room temperature, acidified
with 1 M HCl, and extracted twice with 30 mL of ether. The
organic phase was dried (MgSO4) and concentrated to yield
compound 3 (3.67 g; 100% yield): 1H NMR (CDCl3, 100.3 MHz)
δ 2.62 (t, 2H, CH2Ar), 2.89 (t, 2H, CH2CO), 3.97 (m, 4H, CH2
dioxolane), 5.71 (s, 1H, CH dioxolane), 6.7-7.6 (m, 8H, ArH),
11.0 (s, 1H, COOH).
(ii) When the spacer arm is on position II (linked to
the aromatic ring), most of haptenic determinant groups
remain free, preserving the dibromovinylcyclopropane
part of the molecule, which is characteristic of delta-
methrin. Demoute et al. (1986) used this method to syn-
thesize deltamethrin hapten, which was used in a radio-
immunoassay. The spacer arm was linked on the aro-
matic part of deltamethrin. There were no results on
the sensitivity and specificity of the radioimmunoassay.
The second approach is the use of pyrethric acid to
produce antibodies. The functional group -COOH
allowed a direct coupling of the hapten to the carrier
protein. Pullen and Hock (1995a) conjugated 1(R)-
trans-permethric acid to BSA to produce monoclonal
antibodies for allethrin. The assay was more sensitive
for S-bioallethrin and showed a 43% cross-reactivity
with natural pyrethrins. Bonwick et al. (1994) coupled
permethric acid to the carrier protein through a 6-C
spacer arm to generate polyclonal antibodies against
permethrin. Cross-reactivities were observed for phe-
nothrin, cyfluthrin, and deltamethrin.
In the present work these two approaches were used
to prepare two haptens. We simplified the synthesis of
Demoute et al. (1986) to prepare an analogue of delta-
methrin (hapten 1; Figure 1). Another hapten was
obtained from deltamethric acid (hapten 2; Figure 2).
These two haptens were used to produce monoclonal
antibodies that offer a more definite specificity than
polyclonal antibodies and an unlimited production.
These monoclonal antibodies were used to develop an
immunoassay for the detection of deltamethrin.
3-[4-(3-Formylphenoxy)-1-phenyl]propanoic Acid (4). Five
milliliters of 1 M HCl was added to a solution of compound 3
(0.786 g, 2.5 mmol) in 10 mL of acetone. The mixture was
stirred for 3 h at room temperature and concentrated before
its extraction with ether (2 × 30 mL). The organic layer was
dried over MgSO4 and concentrated to yield 4 (0.675 g; 100%
yield). This compound was pure enough (1H NMR) to be used
directly in the next reaction: 1H NMR (CDCl3, 100.3 MHz) δ
2.69 (t, 2H, CH2-Ar), 2.95 (t, 2H, CH2CO), 6.8-7.6 (m, 8H,
ArH), 11.1 (s, 1H, COOH).
MATERIALS AND METHODS
P r ep a r a tion of Delta m eth r in Ha p ten s. Thin-layer chro-
matography (TLC) and column chromatography were carried
out using Merck silica gel 60. Products 1-5 were identified
by proton NMR spectroscopy with a 100 MHz (J EOL-FX)
instrument. The structures of haptens 1 and 2 were confirmed
by proton NMR spectroscopy with a 400 MHz (Bruker DRX)
(1R-cis)-3-(2,2-Dibromoethenyl)-2,2-dimethylcyclopropane Car-
boxylic Acid Chloride (5). One drop of DMF and 0.32 mL (3.67
mmol) of oxalyl chloride were added to a solution of delta-
methric acid (6) (0.745 g, 2.5 mmol) in 20 mL of dichlo-

1672 J. Agric. Food Chem., Vol. 46, No. 4, 1998
Queffelec et al.
romethane. The reaction mixture was stirred at room tem-
perature for 3 h and dried under vacuum to give the crude
acyl chloride 5, which was pure enough (1H NMR) to be used
directly in the next reaction: 1H NMR (CDCl3, 100.3 MHz) δ
1.27 (s, 3H, CH3), 1.34 (s, 3H, CH3), 2.2 (d, 7.5,1H, CHCO),
2.28 (t, 7.5, 1H, CHCd), 6.51 (d, 7.5, 1H, CHdCBr2).
purified by ultrafiltration (Micro-thin-channel ultrafiltration
system TCF2, Amicon) using a PM 10 000 membrane (Diaflo
ultrafilters, Amicon) and then lyophilized.
Ch a r a cter iza tion of Ha p ten -P r otein Con ju ga tes. The
coupling was assessed according to two methods:
(i) Br elementary analyses were performed by the CNRS
(Service Central d’Analyses) and/or Roussel-UCLAF Co. Hap-
ten density was calculated according to the following formulas,
where n is the number of bromine atoms contained in the
molecule, 560.3 is the deltamethrin contribution to the con-
jugate molecular weight, and 364.8 is the deltamethric acid
contribution:
[1R,1R(R),2R] and [1R,1R(S),2R]-4-[3-{Cyano[(2-(2,2-dibro-
moethenyl)-2,2-dimethylcyclopropyl)carbonyloxy]methyl}phenoxy]-
phenylpropanoic Acid (Hapten 1). Aldehyde 4 (0.675 g, 2.5
mmol) dissolved in 1 mL of toluene was added to a solution of
sodium cyanide (0.265 g, 5.4 mmol) and tetradecyltrimethy-
lammonium bromide (0.0074 g, 0.02 mmol) in 2.3 mL of water
at room temperature under vigorous stirring. The reaction
mixture was stirred for 10 min at room temperature before
acyl chloride 5 (0.79 g, 2.5 mmol) was added. The stirring was
continued for 3 h before a few drops of 1 M HCl was added to
bring the pH to 4. After HCN was removed under vacuum,
partition between ether and water was realized. The organic
layer was dried over MgSO4 and concentrated. The residue
was chromatographed on a silica gel column using ethyl
acetate/hexane 1:3 as eluent to yield a 1-to-1 mixture of
diastereoisomers (0.7 g, 48%). The products were separated
by HPLC using as eluent a mixture of ethyl acetate/hexane/
acetic acid (800:9200:15) with a flow rate of 5 mL min-1. The
retention times of the products were measured: (1R) tR ) 19.13
min, (1S) tR ) 22.44 min, respectively. The absolute config-
uration of CHCN was related to deltamethrin.
BSA-Del
% Br ) 100(n × 79.9)/(BSA weight + n × 0.5 × 560.3)
Ova-Del
% Br ) 100(n × 79.9)/
(ovalbumin weight + n × 0.5 × 560.3)
Ova-DA
% Br ) 100(n × 79.9)/
(ovalbumin weight + n × 0.5 × 364.8)
1R: 1H NMR (CD3OD, 400.1 MHz) δ 1.29-1.31 (2s, 2CH3),
1.91 (d, 8.3, 1H, CHCO), 2.08 (dd, 8.3, 8.4, 1H, CHsCd), 2.70
(t, 7.5, 2H, CH2COOH), 2.97 (t, 7.5, 2H, CH2Ar), 6.32 (s, 1H,
CHCN), 6.69 (d, 8.4, 1H, CHdCBr2), 6.96-7.4 (8H, ArH).
1S: 1H NMR (CD3OD, 400.1 MHz) δ 1.20-1.25 (2s, 2CH3),
1.91 (d, 8.3, 1H, CHCO), 2.08 (dd, 8.3, 8.4, 1H, CHCd), 2.70
(t, 7.5, 2H, CH2COOH), 2.97 (t, 7.5, 2H, CH2Ar), 6.37 (s, 1H,
CHCN), 6.70 (d, 8.4, 1H, CHdCBr2), 6.96-7.4 (8H, ArH).
Elementary analysis: %Ccalcd 43.31, %Cfound 42.56; %Hcalcd
3.98, %Hfound 4.05; %Brcalcd 28.06, %Brfound 27.85.
P r ep a r a tion of Ha p ten 2 Der ived fr om Delta m eth r ic
Acid . Freshly distilled isobutyl chloroformate (0.5 mL, 3.85
mmol) in 3 mL of THF was added at 5 °C to a mixture of
deltamethric acid 6 (0.745 g, 2.5 mmol) and 1.2 mL of
tributylamine in 6 mL of THF. The mixture was stirred for
40 min at 5 °C before 4-aminobutanoic acid ethyl ester
hydrochloride (0.7 g, 4.2 mmol) was added; the stirring was
continued for 1 h at room temperature. THF was removed
under vacuum, and the residue was partitioned between CH2-
Cl2 and H2O. The organic layer was dried over MgSO4, and
after evaporation of the solvent, a crude mixture was obtained.
It was purified on silica gel column chromatography using CH2-
Cl2/EtOAc/AcOH (40:60:1) as eluent to obtain 7 in an oily form.
Compound 7 was diluted with methanol (3 mL) and saponified
with 2.5 mL of 10 M aqueous NaOH while being stirred at
room temperature for 1 h. Twenty milliliters of water and 20
mL of CH2Cl2 were added. The organic layer was discarded.
The aqueous layer was acidified with 3 M aqueous HCl and
extracted twice with CH2Cl2 (2 × 20 mL). The organic extract
was dried over MgSO4 and concentrated under vacuum to yield
hapten 2 as an oil (0.72 g, 75% yield): 1H NMR (CD3OD, 400.1
MHz) δ 1.18, 1.2 (2s, 6H, 2CH3), 1.55 (d, 8.5, 1H, CHCO), 1.79
(t, 8.5, 1H, CHCd), 1.83 (q, 7.1, 2H, CH2N), 2.36 (t, 7.1, 2H,
CH2), 3.25 (q, 7.1, 2H, CH2COOH), 6.5 (t, 7.1, 1H, NH), 6.84
(d, 8.5, 1H, CHdCBr2).
(ii) The level of hapten substitution was estimated by
measuring the loss of free amino groups on the protein.
Protein concentration in conjugate solutions was first mea-
sured according to the method of Bradford (1976). The number
of free amino groups was then determined by a reaction with
orthophthaldialdehyde (OPDA) (Svedas et al., 1980). These
two values were then correlated to estimate the hapten-to-
carrier ratio. OPDA reagent was prepared as follows: 0.5 mL
of OPDA in ethanol (10 mg mL-1) and 0.5 mL of a mercapto-
ethanol/ethanol mixture (0.5:99.5) were added to 30 mL of
borate buffer (0.1 M, pH 9.7). The conjugates were dissolved
in the borate buffer. The conjugate and the reagent were
mixed in a 1-to-1 ratio, and the absorbance of the solution was
measured at 340 nm.
P olyclon a l An tibod y P r od u ction . Female New Zealand
white rabbits were injected subcutaneously four times with 1
mg of conjugate BSA-hapten at 2-week intervals. The first
injection consisted of 0.5 mL of conjugate in a saline solution
(NaCl 8.5‰) emulsified with 0.5 mL of complete Freund’s
adjuvant. For the second injection, incomplete adjuvant was
used instead of complete. Following injections were performed
without adjuvant. The rabbits were bled from the ear vein 2
weeks after each inoculation.
Mon oclon a l An tibod y P r od u ction . Five-week-old female
BALB/c mice were injected intraperitoneally (ip) with 100 µg
of conjugate BSA-hapten at 2-week intervals. The first
injection consisted of 0.25 mL of conjugate in a saline solution
(NaCl 8.5‰) emulsified in 0.25 mL of complete Freund’s
adjuvant. For the second inoculation, incomplete adjuvant was
used instead of complete. The last injections were performed
without adjuvant. Ten days after each inoculation, the mice
were bled from the retroorbital plexus. Three days before the
fusion, 100 µg of immunogen in 500 µL of a saline solution
was injected into the mouse with the highest antiserum titer.
Mouse spleen cells were fused with the Sp2/0-Ag 14 murine
myeloma cells line. The cell mixture was centrifuged (200g,
10 min), and the cell pellet was suspended in poly(ethylene
glycol) warmed at 37 °C (1 mL for 108 spleen cells). After 90
s, 10 mL of RPMI 1640 medium (Gibco) was added dropwise
to dilute the poly(ethylene glycol). The cell suspension was
centrifuged (100g, 10 min), the supernatant was discarded,
and the cell pellet was resuspended in RPMI medium supple-
mented with 10% fœtal calf serum (Myoclone plus, Gibco) and
1% HAT. The cell suspension was dispensed (1 mL/well) into
30 sterile 24-well culture plates. The plates were incubated
at 37 °C in a 5% CO2 atmosphere. Ten to 15 days after the
fusion, the wells containing growing hybridomas in HAT
medium were tested for specific Mabs by ELISA. The HAT
P r epar ation of Hapten -P r otein Con ju gates. The mixed
anhydride method (Erlanger et al., 1959) was used for the
condensation of hapten carboxylic group with carrier amino
group. Isobutyl chloroformate (4.2 µL, 0.032 mmol) was added
dropwise to a cooled solution (12-14 °C) of hapten (0.032
mmol) and tributylamine (9.8 µL, 0.041 mmol) in 0.5 mL of
dry dioxane. The mixture was stirred at the same temperature
for 45 min, and the resulting mixed anhydride solution was
added dropwise to the solution of protein [BSA, 54 mg to
prepare the immunoconjugate or ovalbumin (Ova), 108 mg for
the coating antigen] in 1.5 mL of water/dioxane (5:1, v/v) at
pH 7.5. The mixture was stirred at 4 °C for 5 h while the pH
was maintained at 7.5 with 1 M NaOH. Conjugates were

ELISA for Deltamethrin
J. Agric. Food Chem., Vol. 46, No. 4, 1998 1673
medium was replaced with HT medium. Hybridoma cells from
wells showing a positive response were subcloned by limiting
dilution (Goding, 1980), and clones were further selected by
ELISA.
Mon oclon a l An tibod y Isotyp e. Isotype was determined
by ELISA using affinity-purified goat anti-mouse isotype
antibodies conjugated to alkaline phosphatase (Southern Bio-
technology Associates).
Hapten -Im m obilized Im m u n oassay. Microtitration plates
(Nunc Maxisorp C96) were coated with 100 µL of the Ova-DA
(5 µg mL-1) or Ova-Del (12.5 µg mL-1) conjugate dissolved in
phosphate-buffered saline (pH 7.4) (PBS: NaCl, 0.137 M; KCl,
0.0027 M; KH2PO4, 0.0015 M; Na2HPO4‚12H2O, 0.008 M),
which was equivalent to 1 µg of Ova-DA or 2.5 µg of Ova-Del
coating conjugate per well. The plates were incubated over-
night at 4 °C. Unbound antigen was removed by washing
three times with PBS supplemented with 0.05% Tween 20
(PBS-T). Unspecified binding sites were blocked by adding
250 µL of 0.5% (w/v) fish gelatin solution (Sigma, g-7765) in
PBS (PBS-G) and incubated for 2 h at 25 °C. The blocking
solution was removed, and various concentrations of the
supernatants from hybridoma cultures or serum were screened
for the presence of specific antibodies. Antibodies were diluted
in a PBS-G solution. After incubation at 25 °C for 2 h, the
plates were washed as previously described. The goat anti-
mouse IgG-alkaline phosphatase conjugate (J ackson Immu-
noResearch) diluted in PBS-G (1/2000) was added and incu-
bated at 25 °C for 1 h. After the plates were washed, 100 µL
of the p-nitrophenyl phosphate substrate (1 mg/mL) in dietha-
nolamine buffer (diethanolamine, 1 M; MgCl2, 5 × 10-7 M; pH
9.8) was added and color developed for 1 h. Absorbance of each
well was measured at 405 nm with a microtiter plate scanner
(Multiscan MCC 340 MK, Flow Laboratories).
Com p etitive En zym e-Lin k ed Im m u n osor ben t Assa y.
A precise amount of Ova-Da or Ova-Del was deposed in the
well. After incubation overnight at 4 °C, plates were washed
and blocked with PBS-G for 2 h at 25 °C. After washing, 50
µL/well of anti-deltamethrin antibody and 50 µL/well of
competitor were added to the wells and incubated for 2 h under
stirring. The competitors, dissolved in methanol, were diluted
in PBS-G buffer. The final concentration of methanol in the
well was 3%. After washing, coloration was performed as
described above. The results are expressed in percent inhibi-
tion as follows: % inhibition ) 100 × [1 - (A/Acontrol)].
F igu r e 3. Determination of serum titer of rabbits immunized
with BSA-DA (open symbols) or BSA-Del (solid symbols) with
two coating antigens, Ova-DA (circles) and Ova-Del (squares).
compound 4. The cyanhydrine was obtained by a phase
transfer reaction using sodium cyanide. This cyanhy-
drine was acylated by deltamethric acid chloride to yield
a 1-to-1 mixture of 1R(S) and 1R(R) isomers. The
isomers were separated by semipreparative HPLC to
yield hapten 1.
Another preparation of deltamethrin was developed
by Skerritt and Lee (1996). They used a simple two-
step reaction in conserving the stereochemistry; delta-
methrin alcohol, prepared as described by Zhang and
Scott (1994), was esterified with succinic acid. The
limitation of this synthesis was the poor yield (4%) of
the deltamethrin alcohol.
Another hapten was synthesized by using a delta-
methrin metabolite, that is, the deltamethric acid (Fig-
ure 2). To increase hapten determinant exposure (J ung
et al., 1989), deltamethric acid hapten was obtained by
adding a 4-C spacer arm. This was done with 4-amino-
butanoic acid ethyl ester using isobutyl chloroformate
according to the mixed anhydride method. The result-
ing ester was then saponified to yield hapten 2.
Ch a r a cter iza tion of Con ju ga tes a n d Im m u n iza -
tion . Haptens 1 and 2 were coupled to BSA (immuno-
conjugates) or ovalbumin (coating antigen) by using the
mixed anhydride method (Erlanger et al., 1959). As-
sessing the hapten-to-carrier protein molar ratio was
required before immunization was carried out. Good
antibody titers are generally obtained by using conju-
gates consisting of 8-25 haptens per mole of carrier
(Erlanger, 1980; Wittmann and Hock, 1991). Hapten
protein conjugation was verified by Br elementary
analyses and by measuring the number of free amino
group on the protein by OPDA reaction. The two
methods gave nearly similar results. Hapten densities
calculated from OPDA reaction and from elementary
analysis were, respectively, BSA-Del, 21 and 19; BSA-
DA, 31 and 29; Ova-Del, 10 and 14; and Ova-DA, 15
and 15. These hapten densities were enough, and the
two immunoconjugates (BSA-Del and BSA-DA) were
used for rabbit or mouse immunization.
RESULTS AND DISCUSSION
Ha p ten Syn th esis. Deltamethrin has eight possible
stereoisomers, but the cis-1R,3R configuration about the
cyclopropane ring and the S configuration for the cyano
group at the benzylic carbon are essential for insecticidal
activity (Elliot, 1976). Since the specificity of an im-
munoassay depends on the stereochemistry of the
hapten (Skerritt and Lee, 1996), we have chosen to
maintain hapten stereochemistry during the synthesis.
Demoute et al. (1986) first described the preparation of
deltamethrin hapten in conserving the stereochemistry.
The spacer arm was introduced on the phenoxybenzyl
part of the molecule by using a dialdehyde derivative
and a Horner-Emmons olefination under the conditions
proposed by Masamune (Blanchette et al., 1984). The
subsequent hydrogenation of the activated double bond
yielded compound 4. In the present work we developed
a simplified approach to obtain compound 4 (Figure 1).
The spacer arm was introduced before Ulmann conden-
sation (Lindley, 1984). 3-Bromobenzaldehyde, protected
by a dioxolane, was conjugated to a phenol that bears
the spacer arm 3-(4-hydroxy-1-phenyl)propanoic. Ul-
mann condensation was then achieved under the condi-
tions of Demoute et al. (1986). The conditions of the
reaction (8 h, 200 °C) explained the low yield (46%).
Saponification and deprotection of the aldehyde yielded
P olyclon a l An tibod y Titr a tion . Polyclonal rabbit
antibodies raised against BSA-DA (antiserum 1) and
BSA-Del (antiserum 2) were tested using an indirect
ELISA with the two conjugates Ova-Del and Ova-DA
(Figure 3) (ovalbumin prevents the detection of anti-
BSA antibodies). Antibodies from antiserum 1 bound

1674 J. Agric. Food Chem., Vol. 46, No. 4, 1998
Queffelec et al.
more to Ova-DA than to Ova-Del. The serum titer, that
is, the serum dilution that gave a 3 times the back-
ground absorbance, was 1:100 000 with Ova-Del and
1:250 000 with Ova-DA. Deltamethric acid was the
immunizing hapten, so antibodies were produced against
this molecule. The lower response against Ova-Del,
which still contains the deltamethric acid epitope, could
be explained by the steric bulk of the whole molecule
in which deltamethric acid would be less accessible to
antibodies. On the other hand, antibodies from anti-
serum 2 gave a nearly similar response with the two
conjugates tested: the serum titer was 1:100 000 with
Ova-Del and 1:150 000 with Ova-DA. The greatest
response with Ova-DA indicates that more antibodies
were produced against deltamethric acid epitope than
against the other parts of the whole molecule. This is
in agreement with immunoconjugate structure (BSA-
Del) in which deltamethric acid is the most exposed part
(Figure 1). Skerritt and Lee (1996) observed also that
serum titer was higher when they used the half mol-
ecule to synthesize the hapten rather than the whole
molecule. They explained these results by esterase
hydrolysis of the entire molecule before recognition by
the immune system of the animal. Since the obtained
polyclonal antibodies presented a correct response, the
same immunoconjugates were also used for mice im-
munization. Polyclonal antibodies were also used to
optimizate an ELISA for monoclonal antibody selection.
Hybr id om a P r od u ction . Spleen cells, from mice
exhibiting the highest antiserum titer after immuniza-
tion with BSA-Del or BSA-DA, were fused with the
myeloma cell line Sp2/O-Ag/14. The resulting hybrido-
mas were cultured in 24-well microculture plates. Ten
days after the fusion, growing hybridomas, which were
observed in 90% of the wells, were screened for pyreth-
roids antibodies. The culture supernatants were tested
by indirect ELISA against Ova-Del and Ova-DA. Hy-
bridomas obtained from mice immunized with BSA-DA
presented low responses with the two coating antigens.
They were not used for further investigations. Among
the 594 wells with growing hybridomas obtained from
a mouse immunized with BSA-Del, 30 wells gave a
positive response against Ova-Del and/or Ova-DA. They
were expanded and tested against Ova-Del and/or Ova-
DA, ovalbumin, and BSA. None of them recognized
BSA itself. Eighteen wells were eliminated since they
presented cross-reactivity with ovalbumin, so they were
not specific for the hapten only. Twelve wells contained
an antibody that recognized only the conjugate but not
ovalbumin. Hybridomas from these 12 wells were
cloned by limiting dilution. Wells presenting growing
monoclonal hybridomas were selected by ELISA, as
described above, allowing the isolation of 12 monoclonal
hybridoma cultures.
Mon oclon a l An tibod y Sp ecificity. The 12 mono-
clonal antibodies, named Del 01-Del 12 were IgG1 with
kappa light chain. From their response in indirect
ELISA against Ova-Del and Ova-DA, they were as-
sumed to be distributed into three groups. The first one
(Del 05, Del 08, Del 11) gave an identical response with
Ova-Del and Ova-DA (Figure 4A); this showed that
these antibodies were able to recognize the dibromovi-
nylcyclopropane moiety. The second group (Del 04, Del
07, Del 09, Del 10, Del 12) was more specific to Ova-DA
than to Ova-Del (Figure 4B). The epitope would contain
a common part of deltamethrin and deltamethric acid.
The difference observed in the intensity of the response
F igu r e 4. Determination of monoclonal antibodies specificity
with two coating antigens, Ova-DA (circles) and Ova-Del
(squares).
could be due to deltamethrin steric bulk. The last group
(Del 01, Del 02, Del 03, Del 06) recognized more
effectively Ova-Del than it did Ova-DA (Figure 4C),
indicating that the epitope may include the cyclopropane
ring and the phenoxybenzyl moiety.
Competitive ELISA was performed to evaluate the
ability of these 12 antibodies to detect unconjugated
deltamethrin. Nine monoclonal antibodies presented no
competition with free deltamethrin in the conditions of
the assay; the three others, Del 01, Del 02, and Del 03,
showed a significant competition in the presence of free
deltamethrin.
Assa y Develop m en t. The sensitivity of the assay
depends on both the coating antigen amount and the

ELISA for Deltamethrin
J. Agric. Food Chem., Vol. 46, No. 4, 1998 1675
clonal antibody would recognize the cyclopropane moi-
ety, the halogen on the bound ethylenic, and the cyano
group of deltamethrin.
Con clu sion . To develop an immunoassay for delta-
methrin detection, two haptens were synthesized and
monoclonal antibodies were produced. Hapten 1 had
the same stereochemistry as deltamethrin: 1R cis RS.
It was obtained by introducing a spacer arm on the
phenoxybenzyl group of the molecule. Hapten 2 was
synthesized using deltamethric acid. Although the two
haptens allowed the production of anti-deltamethrin
polyclonal antibodies with high titer, only the BSA-Del
immunoconjugate led to the production of monoclonal
antibodies in the present work. According to their
recognition on Ova-Del and Ova-DA, monoclonal anti-
bodies were classified into three groups. Only those
presenting a better recognition of Ova-Del than of Ova-
DA were effective for the detection of free deltamethrin
in a competitive ELISA. Among these monoclonal
antibodies, Del 01 appeared to be the most sensitive in
competitive ELISA: it allows the detection of delta-
methrin, dissolved in buffer with 3% methanol, in a
range from 0.080 to 2 µg mL-1. Although this detection
limit has to be lowered in an optimized assay, the use
of monoclonal antibody allows the development of a
specific ELISA for deltamethrin detection. Indeed, the
most interesting aspect of monoclonal antibody Del 01
is its specificity to deltamethrin; it has no cross-
reactivity with other pyrethroid molecules, so it could
be used to develop a specific ELISA for deltamethrin.
F igu r e 5. Competitive ELISA for deltamethrin analysis with
the monoclonal antibody Del 01. The assay consisted of 50 µL
of competitor solution and 50 µL of antibody solution.
antibody concentration. These parameters were opti-
mized for the monoclonal antibody Del 01, which
presented the strongest competition in the presence of
free deltamethrin. The optimal ELISA for Del 01 was
performed with 0.133 µg mL-1 monoclonal antibody,
purified by chromatography on protein A (Ey et al.,
1978), and 0.3 µg mL-1 coating antigen Ova-Del. Del-
tamethrin solutions were prepared before each assay
by diluting in PBS-G a freshly prepared methanolic
solution of deltamethrin. The final methanol concentra-
tion in the wells was 3%. Figure 5 shows deltamethrin
standard curve inhibition obtained by the competitive
ELISA. The IC50 value for deltamethrin was estimated
to be 0.5 µg mL-1. The linear range of the assay system
was extended from 0.08 to 2 µg mL-1. Assay sensitivity
was lower than those developed with monoclonal anti-
bodies against other pyrethroids such as permethrin
(Stanker et al., 1989) or allethrin (Pullen and Hock,
1995a). Concerning deltamethrin, results of immunoas-
says developed by Skerritt and Lee (1996) and Demoute
et al. (1986) were unpublished. Skerritt and Lee (1996)
reported only that antibodies, prepared using delta-
methrin hemisuccinate, yielded relatively low sensitivity
assays. Their most sensitive assay used a heterologous
system with antibodies produced from a deltamethrin
hapten coupled on the halogenovinyl group and a
deltamethrin hemisuccinate hapten coupled to an en-
zyme. They obtained a detection limit of ∼0.02 µg mL-1
with freshly prepared deltamethrin.
Cr oss-R ea ct ivit y. To determine the specificity of
monoclonal antibody Del 01, a competitive ELISA was
performed with several pyrethroids (alphamethrin,
bifenthrin, λ-cyalothrin, permethrin, fenvalerate, biores-
methrin). No cross-reactivity was observed, and each
IC50 value was greater than the highest concentration
of pyrethroid used (50 µg mL-1); this corresponded to
<1% cross-reactivity. Therefore, our immunoassay with
the monoclonal antibody Del 01 seems to be specific to
deltamethrin. These results give information about the
epitope recognized by the monoclonal antibody Del 01.
If the specificity of Del 01 was based only on the
R-cyanophenoxybenzyl part of the molecule, λ-cyalo-
thrin, fenvalerate, and cypermethrin would present
cross-reactivity. Since fenvalerate showed no cross-
reactivity, the presence of the halogenovinylcyclopro-
pane part of the molecule is important in antibody
recognition. These observations suggest that the mono-
ABBREVIATIONS USED
BSA, bovine serum albumin; BSA-DA, BSA conju-
gated to hapten 2; BSA-Del, BSA coupled to hapten 1;
DMF, dimethylformamide; ELISA, enzyme-linked im-
munosorbent assay; HAT, hypoxanthine (5 × 10-3 M),
aminopterin (2 × 10-5 M), thymidine (8 × 10-4 M); HT,
hypoxanthine (5 × 10-3 M), thymidine (8 × 10-4 M);
IC50, value of the concentration inhibiting 50% of an
ELISA absorbance value; Mab, monoclonal antibody; 1H
NMR, proton nuclear magnetic resonance; Ova-DA,
ovalbumin coupled to hapten 2; Ova-Del, BSA coupled
to hapten 1; PBS, phosphate-buffered saline; PBS-T,
phosphate-buffered saline plus 0.05% Tween 20; PBS-
G, phosphate-buffered saline plus 0.5% fish gelatin;
THF, tetrahydrofuran.
ACKNOWLEDGMENT
Mr. J . P. Demoute and G. Touyer (Roussel UCLAF
Co., Romainville, France) are warmly thanked for
helpful discussions and for generously providing a
sample of deltamethric acid.
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Received for review May 13, 1997. Revised manuscript received
October 31, 1997. Accepted J anuary 7, 1998. Financial and
material supports from the Conseil Re´gional de Bretagne and
Roussel UCLAF Co. are gratefully acknowledged.
J F9703922