Hapten Synthesis and Production of Monoclonal Antibodies to DDT and Related Compounds

Article abstract of DOI:10.1021/jf9704219

This work describes the production and characterization of monoclonal antibodies (MAbs) to the organochlorine insecticide DDT and their incorporation into several ELISA configurations. A collection of DDT haptens was synthesized by introducing appropriate spacers at two sites of the analyte molecular structure. From mice immunized with hapten-protein conjugates, MAbs with I₅₀ values to p,p'-DDT in the 2-11 nM range in homologous conjugate-coated assays were obtained. According to their cross-reactivity pattern with DDT isomers and metabolites, MAbs can be classified as class-specific or DDT-specific antibodies. Both types of MAbs were obtained from mice immunized with the same hapten-protein conjugate simply by applying a different selection criterion in the screening of fusion supernatants. These immunoassays are potentially very valuable analytical tools for the rapid and sensitive determination of DDT and congeners in food and the environment and for monitoring human exposure to these ubiquitous and toxic compounds.

Full text of DOI:10.1021/jf9704219

3694
J. Agric. Food Chem. 1997, 45, 3694−3702
Ha p ten Syn th esis a n d P r od u ction of Mon oclon a l An tibod ies to DDT
a n d Rela ted Com p ou n d s
Antonio Abad,† J uan J . Manclu´s,† Fatemeh Mojarrad,‡ J osep V. Mercader,† Miguel A. Miranda,‡
J aime Primo,‡ Vicente Guardiola,§ and Angel Montoya*,†
Laboratorio Integrado de Bioingenier´ıa, Universidad Polite´cnica de Valencia, Camino de Vera s/n,
46022 Valencia, Spain, Instituto de Tecnolog´ıa Qu´ımica, UPV-CSIC, Camino de Vera s/n,
46022 Valencia, Spain, and AINIA, Vale`ncia Parc Tecnolo`gic, s/n 46980 Valencia, Spain
This work describes the production and characterization of monoclonal antibodies (MAbs) to the
organochlorine insecticide DDT and their incorporation into several ELISA configurations.
A
collection of DDT haptens was synthesized by introducing appropriate spacers at two sites of the
analyte molecular structure. From mice immunized with hapten-protein conjugates, MAbs with
I
50 values to p,p′-DDT in the 2-11 nM range in homologous conjugate-coated assays were obtained.
According to their cross-reactivity pattern with DDT isomers and metabolites, MAbs can be classified
as class-specific or DDT-specific antibodies. Both types of MAbs were obtained from mice immunized
with the same hapten-protein conjugate simply by applying a different selection criterion in the
screening of fusion supernatants. These immunoassays are potentially very valuable analytical
tools for the rapid and sensitive determination of DDT and congeners in food and the environment
and for monitoring human exposure to these ubiquitous and toxic compounds.
Keyw or d s: ELISA; insecticide; organochlorine pesticide; monoclonal antibodies; DDT; DDE; DDD;
hapten design; hapten heterology; ELISA format
INTRODUCTION
Kennish and Ruppel, 1996). Therefore, an analytical
method for DDT should be able to determine the six
isomers, although method efficiency is normally as-
sessed by the recovery of the most abundant and
frequently encountered p,p′-isomers.
Since the discovery in the 1940s of the insecticidal
properties of the chlorinated compound DDT [dichlo-
rodiphenyltrichloroethane or 1,1,1-trichloro-2,2-bis(p-
chlorophenyl)ethane], more than 1 million tons of this
pesticide have been used worldwide to control insect
pests in agriculture, forestry, and human health (Buser
and Mu¨ller, 1995). DDT is a highly hydrophobic com-
pound with a great stability to physical, chemical, and
biological degradation, which has resulted in the ac-
cumulation of its residues in the adipose tissues of
animals and man, as well as in the environment
(Kennish and Ruppel, 1996). The widespread occur-
rence of DDT, together with the fact that its elimination
from the environment may take decades due to its
persistence, evoked a great concern regarding the
potential toxicological effects of its long-term residues.
As a consequence, the use of DDT was totally banned
in developed countries in the 1970s. Nevertheless, DDT
is still extensively used in developing countries, where
it is very valuable in malaria control programs (Mukher-
jee and Gopal, 1993; Lo´pez-Carrillo et al., 1996; Wal-
iszewski et al., 1996).
Monitoring human and animal exposure to DDT
residues through the analysis of biological tissues and
fluids, such as milk, urine, and blood, is of great
toxicological significance, particularly for those countries
in which DDT is still in use. Furthermore, contamina-
tion of food and the environment by persistent orga-
nochlorine pesticides is a serious concern in tropical
countries (Waliszewski et al., 1996). Traditional meth-
ods for determination of chlorinated pesticides in tissue
samples involve solvent extraction, partitioning of the
pesticide residues from fat into another liquid, and
cleanup of the extract to remove coextractives prior to
gas chromatography. Consequently, the methods are
lengthy and labor-intensive and are therefore not suit-
able for the analysis of the large number of samples
required for comprehensive studies (Ahmad and Marolt,
1986; Herna´ndez et al., 1993; Bucholski et al., 1996;
Menezes and Felix, 1996).
Immunochemical techniques have lately gained a
position as alternative and/or complementary methods
for the analysis of agrochemicals because of their
simplicity, cost-effectiveness, and high sample through-
put. Moreover, immunoassays are field-portable and do
not require sophisticated instrumentation. All of these
features convert immunoassays into very valuable
methods for large monitoring programs (Sherry, 1992;
Van Emon and Lo´pez-AÄ vila, 1992; Meulenberg et al.,
1995; Aga and Thurman, 1997).
In technical formulations, DDT exists in two isomeric
forms, p,p′-DDT and o,p′-DDT, even though the first one
is the only isomer with insecticidal properties. The
main DDT metabolites DDE (dichlorodiphenyldichloro-
ethene) and DDD (dichlorodiphenyltrichloroethane),
which are also lipophilic and even more stable than
DDT, can also exist in the two corresponding isomeric
forms (Buser and Mu¨ller, 1995; Guardino et al., 1996;
The key component of an immunoassay is the anti-
body, since it is responsible for the sensitive and specific
recognition of the analyte. Several attempts to obtain
antibodies for DDT and related compounds have been
previously carried out. Initial work using protein
conjugates of DDA [bis(p-chlorophenyl)acetic acid] as
* Author to whom correspondence should be ad-
dressed (telephone 34-6-3877093; fax 34-6-3877093;
e-mail amontoya@upvnet.upv.es).
† Laboratorio Integrado de Bioingenier´ıa.
‡ Instituto de Tecnolog´ıa Qu´ımica.
§ AINIA.
S0021-8561(97)00421-4 CCC: $14.00
© 1997 American Chemical Society

Monoclonal Antibodies to DDT
J. Agric. Food Chem., Vol. 45, No. 9, 1997 3695
F igu r e 1. Chemical structures of p,p′-DDT and the haptens synthesized for this study.
obtained from Dako (Glostrup, Denmark). DMEM culture
immunogens resulted in low-affinity antisera that mainly
recognized DDA, the chief urinary metabolite of DDT
(Haas and Guardia, 1968; Centeno et al., 1970; Furuya
and Urasawa, 1981; Banerjee, 1987). Further work by
Bu¨rgisser et al. (1990) using a new immunogenic dicofol-
derived hapten allowed them to obtain a monoclonal
antibody (MAb) to DDT that exhibited an I50 around 100
nM in a competitive solid-phase radioimmunoassay.
However, this assay has not found application for
residue analysis probably due to its low sensitivity and
the use of a radioactive label. Immunosystems also
included in its catalog a qualitative/semiquantitative
immunoassay for DDT, but the company has ceased
operation. Therefore, to our knowledge no suitable
enzyme immunoassay for this important and ubiquitous
chemical has been reported. This situation greatly
contrasts with the large number of pesticides for which
enzyme-linked immunosorbent assays (ELISA) have
been developed (Meulenberg, 1995). The aim of this
work was to obtain high-affinity monoclonal antibodies
to DDT and to develop enzyme immunoassays as an
alternative to chromatographic techniques for the screen-
ing of large numbers of biological and environmental
samples.
Hapten design is a key step in the development of
immunoassays for small molecules because the hapten
is primarily responsible for determining antibody rec-
ognition properties (Hammock et al., 1990; Harrison et
al., 1991). To explore the maximum number of pos-
sibilities to obtain good quality antibodies, as well as
for further assay improvement, the synthesis of a
number of haptens with different spacer arms attached
through different molecular sites is generally recom-
mended (Harrison et al., 1990; Goodrow et al., 1995).
Herein, the synthesis of several haptens and their use
to produce MAbs to DDT is described. The character-
ization 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 and its derivatives.
medium, Myoclone Super Plus fetal calf serum, and supple-
ments were from Gibco BRL (Paisley, Scotland). Culture
plasticware was from Bibby Sterilin Ltd. (Stone, U.K.). P3-
X63-Ag8.653 mouse plasmacytoma line was from American
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 Dynat-
ech (Sussex, U.K.), and absorbances were read in dual-
wavelength mode (490-630 nm) with an Emax microplate
reader from Molecular Devices (Sunnyvale, CA). 1H and 13C
NMR spectra were obtained with a Varian VXR-400S spec-
trometer (Sunnyvale, CA), operating at 400 MHz for 1H and
at 100 MHz for 13C. Chemical shifts are reported relative to
tetramethylsilane. UV-vis spectra were recorded on a UV-
160A Shimadzu spectrophotometer (Kyoto, J apan).
Ha p ten Syn th esis. DDT haptens used in this work
(Figure 1) were prepared by introduction of alkyl chain spacers,
ending in a carboxylic acid, in either the central carbon atom
(haptens 1-3) or the aromatic ring (hapten 5) of the DDT
structure. Hapten 4 is only half the complete DDT molecule,
and it was a byproduct in the synthesis of hapten 1.
5,5-Bis(4-chlorophenyl)-5-hydroxypentanoic Acid (1) and
5-(4-Chlorophenyl)-5-oxopentanoic Acid (4, Figure 2). Glutaric
anhydride (1.14 g, 0.01 mol) was placed in dry ether (40 mL)
in the presence of argon. After warming to dissolve the
anhydride, p-chlorophenylmagnesium bromide (0.03 mol, 30
mL of a 1 M ether solution) was added from a dropping funnel.
The reaction mixture was boiled under reflux for 2 h and
finally was stirred at room temperature for 14 h. After this
time, the reaction mixture was poured over a saturated
solution of ammonium chloride. The aqueous phase was
washed with ether, acidified with 1 M HCl (1:1 by volume),
and extracted with ether. The organic phase was washed with
water, dried (MgSO4), and concentrated. The weight of residue
was 2.5 g. Hapten 4, previously described by Masse et
al.(1989), was obtained upon recrystallization from water (1.3
g, 50%), while hapten 1 remained undissolved (1 g, 40%).
Hapten 4 formed white crystals: mp 122-124 °C (water);
1H NMR (CDCl3) δ 7.9 (d, J ) 8 Hz, 2H, 2,6-ArH), 7.43 (d, J
) 8 Hz, 2H, 3,5-Ar-H), 3.05 (t, J ) 7 Hz, 2H, ArCOCH2), 2.50
(t, 2H, J ) 7 Hz, CH2COOH), 2.07 (t, J ) 7 Hz, 2H, CH2CH2-
CH2); 13C NMR 198.2 (CO, ketone), 179.1 (CO, acid), 139.7
(CCl), 135.1 (CCO), 129.5 and 129 (aromatic CH), 37.2, 32.8,
and 18.8 (CH2).
MATERIALS AND METHODS
Ch em ica ls, Im m u n or ea gen ts, a n d In str u m en ts. Stan-
dards of p,p′-DDT, o,p′-DDT, p,p′-DDE, o,p′-DDE, p,p′-DDD,
o,p′-DDD, and dicofol were from Riedel-de Hae¨n (Seelze,
Germany). Stock solutions were prepared in dry DMF and
stored at 4 °C. Starting products for hapten synthesis and
hapten-protein coupling reagents were obtained from Fluka-
Aldrich Qu´ımica (Madrid, Spain).
Freund’s adjuvants, OVA, and OPD were obtained from
Sigma Qu´ımica (Madrid, Spain). BSA fraction V, enzyme
immunoassay grade HRP, Hybridoma Fusion and Cloning
Supplement (HFCS), and PEG 1500 were purchased from
Boehringer Mannheim (Germany). Peroxidase-labeled rabbit
anti-mouse Igs and affinity-isolated goat anti-mouse Igs were
Hapten 1 was an oily yellowish compound: 1H NMR (CDCl3)
δ 7.24-7.22 (m, 4H aromatic), 2.40 (t, J ) 7 Hz, 2H,
CH2COOH), 2.27 [m, 2H, C(OH)CH2], 1.57-1.52 (m, 2H,
CH2CH2CH2); 13C NMR 170.7 (CO), 141.9 and 133.9 (aromatic
C), 128.9 and 127.0 (aromatic CH), 86.9 (COH), 33.1, 29.0, and
16.5 (CH2).
1,1-Bis(4-chlorophenyl)-2,2,2-trichloroethylaminocarbonyl-
acetic Acid (2, Figure 3). Dicofol (250 mg, 0.67 mmol), ethyl
cyanoacetate (1 mL), and acetic acid (1 mL) were placed
together in a round-bottom flask, and 96% sulfuric acid (4 mL)
was added slowly. After the addition was complete, the
mixture was warmed over a hot water bath at 50 °C for 15
min. The reaction mixture became dark red, and then it was

3696 J. Agric. Food Chem., Vol. 45, No. 9, 1997
Abad et al.
F igu r e 2. Reaction scheme of the synthesis of haptens 1 and 4.
F igu r e 3. Reaction scheme of the synthesis of hapten 2.
F igu r e 4. Reaction scheme of the synthesis of hapten 3.
stirred at room temperature for 20 h. The mixture was poured
over water and extracted with ether. The ether phase was
washed with water and sodium bicarbonate solution, dried,
and concentrated. The crude product (200 mg) was purified
on a silica gel preparative plate using hexane/CH2Cl2 (1:1) as
eluant. Three fractions were separated. Dicofol was recovered
(60 mg), the second compound was a dimeric ether of dicofol
(70 mg), and the least polar compound was the ethyl ester of
hapten 2 (30 mg): 1H NMR (CDCl3) δ 9.15 (m, 1H, NH), 7.55
(d, J ) 8 Hz, 4H aromatic), 7.29 (d, J ) 8 Hz, 4H aromatic),
4.29 (q, J ) 7 Hz, 2H, CH2CH3), 3.38 (s, 2H, COCH2), 1.33 (t,
J ) 7 Hz, 3H, CH2CH3).
The ester was hydrolyzed to acid using sodium ethanolate.
Sodium (6 mg) was dropped into ethyl alcohol (1 mL) in a small
flask provided with a condenser; the mixture was heated until
all of the sodium was dissolved and then cooled in an ice-salt
bath. Then the ester (30 mg) was added and the reaction
mixture was stirred for 15 min. Thereafter, the solvent was
evaporated. Water was added, and the mixture was acidified
with dilute HCl and extracted with ether, dried, and concen-
trated to afford 15 mg of hapten 2: 1H NMR (CDCl3) δ 9 (bs,
1H, NH), 7.55 (d, J ) 10 Hz, 4H aromatic), 7.29 (d, J ) 10 Hz,
4H aromatic), 3.43 (s, 2H, CH2); 13C NMR 171.2 (COOH), 163.6
(CONH), 136.3 and 134.7 (aromatic C), 131.5 and 127.6
(aromatic CH), 104.9 (CCl3), 77.4 (CNHCO), 40.55 (CH2).
4,4-Bis(4-chlorophenyl)-3-butenoic Acid (3, Figure 4). This
hapten was synthesized by an alternative route to that
described by Himmele et al. (1985). Succinic anhydride (1.0
g, 0.01 mol) was placed in dry ether (40 mL) in the presence
of argon. After warming to dissolve the anhydride, p-chlo-
rophenylmagnesium bromide (0.03 mol, 30 mL of a 1 M ether
solution) was added from a dropping funnel. The reaction
mixture was boiled under reflux for 2 h and finally stirred at
room temperature for 14 h. After this time, the mixture was
poured over a saturated solution of ammonium chloride. The
aqueous phase was washed with ether, acidified with 1 M HCl
(1:1 by volume), and extracted with ether. The ethereal extract
was washed with water, dried (MgSO4), and concentrated to
afford an oily residue (2.6 g). Purification of 400 mg of this
crude was achieved by esterification with methanol/sulfuric
acid, separation by silica gel preparative chromatography
(hexane/CH2Cl2, 1:1), and hydrolysis. This led to 140 mg of
hapten 3: 1H NMR (CDCl3) δ 7.40-7.09 (m, 8Η, aromatic),
6.22 (t, J ) 8 Hz, 1H, CdCH), 3.19 (d, J ) 8 Hz, 2H, CH2).
4-{4-[1-(4-Chlorophenyl)-2,2,2-trichloroethyl]phenyl}bu-
tanoic Acid (5, Figure 5). Chloral hydrate crystals (2.3 g, 0.002

Monoclonal Antibodies to DDT
J. Agric. Food Chem., Vol. 45, No. 9, 1997 3697
F igu r e 5. Reaction scheme of the synthesis of hapten 5.
mol), chlorobenzene (2 mL, 0.002 mol), and 4-phenylbutanoic
acid (1.6 g, 0.001 mol) were placed together in a round-bottom
flask and warmed on a water bath, with occasional shaking,
until all crystals dissolved. The reaction mixture was cooled
in an ice bath, and 96% sulfuric acid (7 mL, 6.4 g) was added
slowly. The mixture was shaken mechanically for 1.5 h. After
this time, it was poured slowly with stirring into water (100
mL). Then it was extracted with ether, dried, and concen-
trated. The crude product (2.5 g) was purified on silica gel
preparative chromatography plates (200 mg per plate) using
CH2Cl2 as eluant. This way, 1 g of hapten 5 was obtained:
1H NMR (CDCl3) δ 7.49-7.04 (m, 8H, aromatic), 2.47 (bs, 2H,
CH2), 2.25 (bs, 2H, CH2), 1.79 (bs, 2H, CH2); 13C NMR 173
(COOH), 142, 136.7, 135.4 and 133.9 (aromatic C), 131.3, 129.9,
128.4 and 128.2 (aromatic CH), 101.3 (CCl3), 70,34 (CHCCl3),
35, 35.2, and 27 (CH2); IR (CH2Cl2) ν-1 cm-1 1704 (CO).
P r ep a r a tion of Im m u n izin g Con ju ga tes. Haptens 1, 2,
and 5 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-hydroxysuc-
cinimide and dicyclohexylcarbodiimide 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 spectro-
photometrically. UV-vis spectra showed qualitative differ-
ences between carrier proteins 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 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 16, 20, and 23 for haptens 1,
2, and 5, respectively.
gate concentrations and molar ratios were estimated spectro-
photometrically. With the same assumptions as before, the
estimated hapten to enzyme molar ratios were 3, 4, 1, 1, and
10 for haptens 1, 2, 3, 4, and 5, respectively.
P r od u ction of MAbs to DDT. Immunization. BALB/c
female mice (8-10 weeks old) were immunized with BSA-1,
-2, and -5 conjugates. First dose consisted of 30 µg of
conjugate intraperitoneally injected as an emulsion of PBS (10
mM phosphate, 137 mM NaCl, 2.7 mM KCl, pH 7.4) and
complete Freund’s adjuvant. Two subsequent injections with
immunogens emulsified in incomplete Freund’s adjuvant were
given at 3 week intervals. One week after the last injection,
mice were tail-bled and sera tested for antihapten 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 intraperitoneal injection of 100 µg of conjugate in PBS,
4 days prior to cell fusion.
Cell Fusion. P3-X63-Ag8.653 murine myeloma cells (ATCC,
Rockville, MD) were cultured in high-glucose DMEM supple-
mented with 2 mM GLUTAMAX I, 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 × 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
days 4 and 7 postfusion. Cells were grown in HAT medium
for 1 week, and then HAT was substituted by HT medium
(HAT medium without aminopterine).
Hybridoma Selection and Cloning. Eight to eleven days
after cell fusion, culture supernatants were screened for the
presence of antibodies that recognized p,p′-DDT. The screen-
ing consisted of the simultaneous performance of a noncom-
petitive and a competitive indirect ELISA, to test the ability
of antibodies to bind the OVA conjugate of the immunizing
hapten and to recognize p,p′-DDT, 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 (ca. 106 cells/well) and peritoneal macrophages (ca.
5000 cells/well). Stable antibody-producing clones were ex-
panded and cryopreserved in liquid nitrogen.
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-vis spectrophotometry. By assuming
additive absorbance values, hapten to protein molar ratios
were evaluated as 4, 21, 3, 9, and 11 for haptens 1, 2, 3, 4,
and 5, respectively.
Purification of MAbs. Antibodies were purified directly from
late stationary phase culture supernatants by saline precipita-
tion followed by anion-exchange chromatography on DEAE-
Sepharose (Sigma). Most culture supernatants 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.
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 tri-n-butylamine 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 1.8 mL of DMF was added, 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 conju-
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

3698 J. Agric. Food Chem., Vol. 45, No. 9, 1997
Abad et al.
Ta ble 1. Su m m a r y of Cell F u sion a n d Hybr id om a
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 temperature. 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 conjugate-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 for the study of antibody sensitivity and
specificity to DDT. In the antibody-coated format, the specific
antibody was coated directly or by using a capture auxiliary
antibody, and competitive ELISAs were followed to evaluate
the assay properties using different enzyme tracers (HRP-
hapten conjugates).
For competition assays, the concentrations of antibodies,
hapten conjugates, or enzyme tracers were optimized by
checkerboard titration. Competitive curves were obtained by
plotting absorbance against the logarithm of analyte concen-
tration. Sigmoidal curves were fitted to a four-parameter
logistic equation (Raab, 1983), using Sigmaplot software
package (J andel Scientific, Germany).
Conjugate-Coated Format. Plates were coated with 1 µg/
mL of OVA-hapten conjugates. Then, different antibody
concentrations in PBS containing 0.1% BSA were added and
incubated for 2 h. Next, plates were incubated for 1 h with
peroxidase-labeled rabbit anti-mouse Igs 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 the
competitor (p,p′-DDT or related compounds) in PBS 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 DDT standards diluted in PBS
(50 µL/well) and the selected concentrations of enzyme tracers
(50 µL/well). Peroxidase activity was measured as above.
Indirect Antibody-Coated Format. The difference with 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.
Selection Resu lts
no. of wells
no. of
cloned
immunizing fusion
positivea competitiveb
(analyte)
hapten
no. seeded (conjugate)
hybridomasc
1
1
2
1
1
2
3
4
5
288
152
29
1
20
5
36
5
8
1
0
0
1
0
0
-
4
0
2
5
744e
384
d
3
384
288
36
0g
384
4g
-
3
1632f
41
32g
a
Wells with antibodies that recognized the OVA-hapten
conjugates (homologous assays) by indirect ELISA (absorbance >
b
0.5). Wells with antibodies that recognized 1 µM p,p′-DDT in
solution (inhibition > 50%). c Only hybridomas secreting antibodies
with the lowest I50 for p,p′-DDT were stabilized and cloned.
d
Indicates that competitive antibodies were initially cloned, but
they were rejected during the cloning proccess because better
antibodies were obtained in further fusions. e This cell fusion was
carried out with lymphocytes obtained from two spleens. f The
number of seeded wells was significantly higher in this fusion than
in the rest because hybridomas were seeded at a lower cell density
by using HFCS. g In the screening of these cell fusions, wells were
considered competitive if the signal was inhibited >50% by 0.1
µM p,p′-DDT and <25% by 0.1 µM p,p′-DDE.
P r od u ction of An tibod ies to DDT. Mouse Poly-
clonal Response. To evaluate the suitability of the
synthesized haptens to raise anti-DDT antibodies, mice
were immunized with BSA conjugates of haptens 1, 2,
and 5. After three injections, the titer (serum dilution
giving an absorbance around 1.0 in the established
conditions) of antibodies recognizing conjugated haptens
was estimated by indirect ELISA using the respective
homologous OVA-hapten as coating conjugate. Sera
from all of the immunogens showed antihapten reactiv-
ity with titers in the (1-5) × 104 range and were
subsequently tested for their ability to recognize p,p′-
DDT by indirect competitive ELISA. Only sera from
mice immunized with BSA-5 bound competitively p,p′-
DDT, with a concentration producing 50% inhibition of
antibody binding (I50) of 1 µM p,p′-DDT. No inhibition
was found for the rest of the sera.
Production of MAbs. The immunological properties
of mouse polyclonal and monoclonal antibodies raised
to the same haptenic structure are often fairly different.
In fact, MAbs with an affinity to the analyte up to 4
orders of magnitude better than that of the mouse sera
have been obtained (Abad and Montoya, 1994). Then,
although an anti-DDT polyclonal response was demon-
strated only with the BSA-5 immunogen, at least one
cell fusion was undertaken for each immunizing hapten.
Cell fusion and hybridoma selection results are sum-
marized in Table 1. The yield of wells with antibodies
recognizing the corresponding homologous conjugated
haptens (positive clones) was highly variable from one
fusion to another. Moreover, not all of the positive wells
showed reactivity to p,p′-DDT (competitive wells). In
the first fusion carried out from each immunizing
hapten, wells showing inhibition by 1 µM p,p′-DDT were
found only from mice immunized with haptens 1 and 5
(one and three hybridomas, respectively). As the objec-
tive of this work was to obtain high-affinity antibodies
to DDT, additional fusion effort was focused on mice
immunized with haptens that gave positive results in
the first fusion.
RESULTS AND DISCUSSION
Syn th esis of Ha p ten s. Regarding its immunogenic
structure, DDT is a small, highly hydrophobic, and
simple analyte, consisting of two aromatic rings sub-
stituted with a chlorine atom and connected to each
other by a carbon atom (Figure 1). An appropriate
hapten design should preserve as many substituents as
possible and produce minor modifications in the ring
electronic distribution as a consequence of spacer at-
tachment (Manclu´s and Montoya, 1996). Consequently,
two approaches were followed to prepare haptens re-
sembling the DDT chemical structure: (I) attachment
of a spacer arm through the carbon atom that joins the
two aromatic rings and (II) introduction of a spacer arm
in the aromatic ring by substitution of the chlorine atom.
The first strategy led to the synthesis of haptens 1-3,
whereas hapten 5 belongs to the second chemical
approach. Consistent with their highest similarity to
the analyte, compounds 1, 2, and 5 were used as
immunizing and assay haptens, whereas haptens 3 and
4 were used to prepare heterologous conjugates. Het-
erologous conjugates based on haptens consisting of only
a part of the complete analyte structure, such as
compound 4, have been previously used to improve the
sensitivity of immunoassays for PCBs (Chiu et al., 1995)
and chlorpyrifos (Manclu´s et al., 1996).
In fusion 2 with hapten 1, the only positive well did
not contain antibodies showing 50% inhibition by a
concentration cutoff of 1 µM p,p′-DDT. However, the
second fusion from hapten 5 was especially successful,
since all positive wells behaved competitively. In fact,

Monoclonal Antibodies to DDT
J. Agric. Food Chem., Vol. 45, No. 9, 1997 3699
Ta ble 2. Affin ity to p,p′-DDT of MAbs Usin g Hom ologou s
Ha p ten s in th e Con ju ga te-Coa ted (CC) a n d in th e
In d ir ect An tibod y-Coa ted (IAC) F or m a ts
petitive signal >50% by 0.1 µM p,p′-DDT and <25% by
0.1 µM p,p′-DDE were considered competitive. Among
those wells that fulfilled this criterion, the three hybri-
domas displaying the highest inhibition by p,p′-DDT and
the lowest inhibition by p,p′-DDE were selected and
cloned.
Ch a r a cter iza tion of th e MAbs. MAbs obtained
from haptens 1 (LIB1-11) and 5 (LIB5-*) were charac-
terized for affinity and specificity to DDT using homolo-
gous and heterologous haptens in different ELISA
formats.
Affinity. The ability of the eight MAbs to recognize
p,p′-DDT was first determined using the homologous
conjugate-coated ELISA format. I50 values ranged from
2.1 to 11.1 nM (Table 2). All MAbs derived from hapten
5 displayed a higher affinity to p,p′-DDT than the MAb
derived from hapten 1. When the antibodies were
assayed in the same ELISA format but using heterolo-
gous conjugates, LIB1-11 MAb recognized coating con-
jugates of haptens 3 and 4, although competitive assays
with these heterologous conjugates did not improve the
high sensitivity of the homologous assay (I50 values of
39.8 and 12.4 nM with the conjugates OVA-3 and
OVA-4, respectively). None of the MAbs derived from
hapten 5 (LIB5-* MAbs) recognized heterologous con-
jugates based on haptens 1-4, so no competitive het-
erologous assays could be performed. It should be noted
that MAbs derived from hapten 5, with the spacer arm
I50 (nM)
MAb
CC format
IAC format
LIB1-11
LIB5-21
LIB5-25
LIB5-28
LIB5-212
LIB5-51
LIB5-52
LIB5-53
11.1
2.1
2.1
2.3
2.9
4.6
6.4
6.5
90.8
13.0
16.0
13.4
14.1
9.3
11.4
9.4
most of them displayed an affinity to p,p′-DDT higher
than that of the antibodies raised in the first fusion.
Therefore, the three antibodies raised from fusion 1
were discarded and the four hybridomas from fusion 2
showing the strongest recognition of p,p′-DDT were
stabilized and cloned. Preliminary characterization of
these antibodies from culture supernatants indicated
that all of them recognized p,p′-DDE and p,p′-DDT
almost equally. At this point we reasoned that it would
also be of analytical interest to obtain antibodies able
to discriminate between both compounds. With this
idea in mind, new cell fusions were undertaken with
hapten 5. In the screening of fusion supernatants, both
p,p′-DDE and p,p′-DDT were used as competitors, so
only those wells showing an inhibition of the noncom-
Ta ble 3. MAb Recogn ition of DDT Isom er s in th e Hom ologou s Con ju ga te-Coa ted ELISA F or m a ta
a
Coating conjugates were OVA-1 for the LIB1-11 MAb and OVA-5 for the rest of the antibodies. Both conjugates were used at 1
b
µg/mL. Each antibody was used at a concentration previously determined as optimal. Percentage of cross-reactivity ) (I50 of p,p′-DDT/
I50 of other compound) × 100.

3700 J. Agric. Food Chem., Vol. 45, No. 9, 1997
Abad et al.
Ta ble 4. MAb Recogn ition of DDT Isom er s in th e Hom ologou s In d ir ect An tibod y-Coa ted ELISA F or m a ta
a
The goat anti-mouse capture antibody was used at 2 µg/mL, and the MAbs at 1 µg/mL. The enzyme tracer was HRP-5 for all MAbs,
b
and it was used at the concentration previously determined as optimal for each MAb. Percentage of cross-reactivity ) (I50 of p,p′-DDT/
I50 of other compound) × 100.
attached to the aromatic ring, did not recognize conju-
gates from hapten 1, derivatized at the central carbon
atom, and vice versa. Therefore, although hapten
heterology has been demonstrated as a good approach
to improve the sensitivity of pesticide immunoassays
(Wie and Hammock, 1984; Marco et al., 1995), the
heterologous hapten structure and/or the way it is
presented to the antibody should not involve great
differences with respect to the immunizing structure.
Otherwise, haptens could become unrecognized by the
antibody and unsuitable to develop competitive immu-
noassays. In the authors’ experience, suitable heter-
ologous haptens are those prepared by introducing
slight changes in the hapten structure (change or
elimination of a substituent), by changing the type and/
or length of the spacer arm, or by changing the attach-
ment site to a position proximal to that used in the
immunogen (Manclu´s and Montoya, 1996; Manclu´s et
al., 1996; Abad et al., 1997).
The competitive behavior of the MAbs in the direct
and indirect antibody-coated formats was also evalu-
ated. MAbs directly immobilized to the plate at 1 µg/
mL were not able to recognize any of the HRP-hapten
conjugates, so no competitive assay could be performed
with this assay configuration (the maximum concentra-
tion of enzyme conjugate tested was 10 µg/mL). On the
contrary, MAbs immobilized to the plate through a
capture immunoglobulin (indirect antibody-coated for-
mat) recognized their corresponding homologous enzyme
conjugates, while the heterologous ones remained un-
recognized. Therefore, competitive assays were per-
formed with the HRP-1 conjugate for the LIB1-11 MAb
and with the HRP-5 conjugate for the LIB5-* MAbs. For
all of the MAbs, the I50 value obtained in the indirect
antibody-coated format was higher than in the conjugate-
coated format, so less sensitive immunoassays were
obtained (Table 2).
Specificity. The specificity of the MAbs was evaluated
by performing competitive assays with several com-
pounds of the DDT family as competitors, and the
obtained I50 values were used to calculate cross-reac-
tivities. Except for LIB1-11 MAb, antibody cross-
reactivities were investigated in both the conjugate-
coated format (Table 3) and the antibody-coated format
(Table 4). Comparison of the data in Tables 3 and 4
suggests that specificity is mainly determined by the
MAb more than by the assay format used. The only
noticeable change in the MAb cross-reactivity pattern
is a lower recognition of DDD isomers in the indirect
antibody-coated format with respect to the CR values
found in the conjugate-coated format.
According to the recognition pattern exhibited, MAbs
could be classified into three different groups. First,
monoclonal antibodies LIB5-21, -25, -28, and -212
exhibited high cross-reactivity values for all of the
compounds of the DDT family, particularly for the p,p′-
isomers, which are the most relevant from an analytical
point of view. Therefore, these MAbs can be considered

Monoclonal Antibodies to DDT
J. Agric. Food Chem., Vol. 45, No. 9, 1997 3701
as unspecific antibodies or, more appropriately, class-
specific antibodies. Second, monoclonal antibodies LIB5-
51, -52, and -53 were DDT-specific antibodies, since they
recognized almost equally p,p′- and o,p′-DDT isomers,
while displaying low CR values for the rest of com-
pounds (<11%). Third, LIB1-11 MAb displayed a dif-
ferent behavior characterized by CRs of 56.2% to p,p′-
DDD and 5.6-13.9% for the rest of compounds. Not
surprisingly, this MAb was the only one that recognized
dicofol to some extent (CR ) 8.2%), which is in ac-
cordance with the fact that this MAb derives from
hapten 1, and both dicofol and hapten 1 have a hydroxyl
group joined to the central carbon atom. Nevertheless,
the ideal antibody for analytical purposes should be
completely specific for a determined analyte or com-
pletely specific of a certain class of compounds, showing
very similar cross-reactivity values for all of them.
Therefore, the analytical uselfulness of an antibody
displaying an intermediate situation, like LIB1-11 MAb,
is uncertain. This fact, together with the lower affinity
to DDT-related compounds shown by LIB1-11 MAb, led
us to focus our attention on LIB5-* MAbs.
Among class-specific antibodies, LIB5-25 MAb showed
the highest and most homogeneous cross-reactivity
values, so this antibody seems to be the most adequate
to develop an immunoassay for DDT isomers and
metabolites. With respect to the development of an
immunoassay specific for DDT, LIB5-52 and LIB5-53
MAbs displayed the lowest cross-reactivity values for
DDT metabolites along with a similar affinity to DDT
isomers. The choice of LIB5-52 MAb for this purpose
was based on the fact that it can be used at a concentra-
tion 10 times lower than LIB5-53 MAb. Competitive
curves obtained with LIB5-25 and LIB5-52 MAbs in the
conjugate-coated format to DDT isomers and metabo-
lites are presented in Figure 6.
F igu r e 6. Competitive curves in the homologous conjugate-
coated ELISA format obtained with the MAbs LIB5-25 (top)
and LIB5-52 (bottom). The coating conjugate OVA-5 was used
at 1 µg/mL, and the MAbs were used at 0.1 and 0.2 µg/mL
(LIB5-25 and LIB5-52, respectively). Standards in the range
10-4-2 × 103 nM were prepared by serial dilutions in PBS
from the stock solutions in DMF. Points represent the average
of absorbance values from triplicate wells, and they were fitted
to a four-parameter logistic equation (lines). Competitors: p,p′-
DDT (b); o,p′-DDT (O); p,p′-DDE (9); o,p′-DDE (0); p,p′-DDD
(2); o,p′-DDD (1).
CONCLUSIONS
Following general guidelines about hapten design,
several haptens were synthesized and examined for
their ability to produce monoclonal antibodies to the
DDT family of pesticides. Two types of immunizing
haptens, characterized by the presentation of different
parts of the DDT molecular structure, were prepared.
Although only the polyclonal response from hapten 5
showed the presence of anti-DDT antibodies, high-
affinity MAbs were obtained from mice immunized with
both hapten 1 and hapten 5 immunogens (I50 values in
the low nanomolar range). In fact, the affinity to p,p′-
DDT of these MAbs, particularly that of the antibodies
derived from hapten 5, is higher by far than that
exhibited by previously reported antibodies (Bu¨rgisser
et al., 1990) Taking into account the affinity and the
number of MAbs obtained, hapten 5 seems to mimic
better the DDT molecular structure than hapten 1 does.
To our knowledge, this is the first time that a hapten
derivatized at the aromatic ring has been used to
produce antibodies to DDT. All previous attempts to
obtain anti-DDT antibodies used the central carbon
atom as the derivatization site to prepare the hapten.
In the evaluation of the analytical properties of
immunoassays performed by combining several assay
haptens and formats, no improvement was found.
Competitive assays with DDT isomers and metabolites
evidenced that anti-DDT MAbs may be classified into
two main groups according to their cross-reactivity pat-
tern: class-specific antibodies and DDT-specific anti-
bodies. Interestingly, both classes of antibodies were
elicited from the same hapten (5) simply by applying a
different selection criterion in the screening of fusion
supernatants. This fact points out the importance of a
well-designed fusion screening for selecting MAbs with
the desired characteristics.
Two of the MAbs produced were selected for further
studies on the basis of their high affinity and different
specificity to DDT-related compounds. The most sensi-
tive assays with these antibodies, named LIB5-25 and
LIB5-52, were based on the homologous conjugate
OVA-5 in the conjugate-coated ELISA format (I50
values of 2.1 and 6.4 nM, respectively). Work is in
progress to assess the influence of physicochemical
factors on the performance of both immunoassays and
to study their feasibility for the analysis of DDT residues
in biological and environmental samples.
ABBREVIATIONS USED
BSA, bovine serum albumin; CR, cross-reactivity;
DMEM, Dulbecco’s Modified Eagle’s Medium; DDA, bis-
(p-chlorophenyl)acetic acid; DDD, dichlorodiphenyldi-
chloroethane; DDE, dichlorodiphenyldichloroethene;
DDT, dichlorodiphenyltrichloroethane; DMF, N,N-di-

3702 J. Agric. Food Chem., Vol. 45, No. 9, 1997
Abad et al.
ACS Symposium Series 451; American Chemical Society:
Washington, DC, 1990; pp 14-27.
Harrison, R. O.; Goodrow, M. H.; Hammock, B. D. Competitive
inhibition ELISA for the s-triazine herbicides. Assay opti-
mization and antibody characterization. J . Agric. Food
Chem. 1991, 39, 122-128.
methylformamide; ELISA, enzyme-linked immunosor-
bent assay; HFCS, Hybridoma Fusion and Cloning
Supplement; HRP, horseradish peroxidase; I50, concen-
tration giving 50% inhibition of maximum response; Ig,
immunoglobulin; MAb, monoclonal antibody; NMR,
nuclear magnetic resonance; OPD, o-phenylenediamine;
OVA, ovalbumin; PBS, phosphate-buffered saline; PBST,
phosphate-buffered saline containing 0.05% Tween 20;
PEG, poly(ethylene glycol); TLC, thin-layer chromatog-
raphy; UV-vis, ultraviolet-visible.
Herna´ndez, L. M.; Ferna´ndez, M. A.; Hoyas, E.; Gonza´lez, M.
J .; Garc´ıa, J . F. Organochlorine insecticide and polychlori-
nated biphenyl residues in human breast milk in Madrid
(Spain). Bull. Environ. Contam. Toxicol. 1993, 50, 308-315.
Himmele, W.; Hoffmann, W.; J anitschke, L. (BASF A.-G.)
4-Substituted but-3-ene-1-carboxylic acids and their esters.
Ger. Offen. DE 3,345,375 (C1. C07C51/12), 1985.
Kennish, M. J .; Ruppel, B. E. DDT contamination in selected
estuarine and coastal marine finfish and shellfish of New
J ersey. Arch. Environ. Contam. Toxicol. 1996, 31, 256-262.
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.
Lo´pez-Carrillo, L.; Torres-Arreola, L.; Torres-Sa´nchez, L.;
Espinosa-Torres, F.; J ime´nez, C.; Cebria´n, M.; Waliszewski,
S.; Saldate, O. Is DDT use a public health problem in
Mexico? Environ. Health Perspect. 1996, 104, 584-588.
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.
Masse, R.; Messier, F.; Ayotte, C.; Levesque, M. F.; Sylvestre,
M. A comprehensive gas chromatographic/mass spectromet-
ric analysis of 4-chlorobiphenyl bacterial degradation prod-
ucts. Biomed. Environ. Mass Spectrom. 1989, 18, 27-47.
Menezes, M. L.; Felix, G. Analysis of organochlorine pesticides
in plain milk using direct injection on an ISRP column, with
column switching. J . Liq. Chromatogr. Relat. Technol. 1996,
19, 3221-3228.
LITERATURE CITED
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.
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. 1997,
45, 1486-1494.
Aga, D. S.; Thurman, E. M. Environmental inmmunoassays:
alternative techniques for soil and water analysis. In
Immunochemical Technology for Environmental Applica-
tions; Aga, D. S., Thurman, E. M., Eds.; ACS Symposium
Series 657; American Chemical Society: Washington, DC,
1997; pp 1-20.
Ahmad, N.; Marolt, R. S. One-step extraction and cleanup
procedure for determination of p,p′-DDT, p,p′-DDD, and p,p′-
DDE in fish. J . Assoc. Off. Anal. Chem. 1986, 69, 581-586.
Banerjee, B. D. Development of an enzyme-linked immun-
osorbent assay for the quantification of DDA [2,2-bis-(4-
chlorophenyl)acetic acid] in urine. Bull. Environ. Contam.
Toxicol. 1987, 38, 798-804.
Bucholski, K. A.; Begerow, J .; Winneke, G.; Dunemann, L.
Determination of polychlorinated biphenyls and chlorinated
pesticides in human and body fluids and tissues. J . Chro-
matogr. A 1996, 754, 479-485.
Bu¨rgisser, D.; Frey, S.; Gutte, B.; Klauser, S. Preparation and
characterization of polyclonal and monoclonal antibodies
against the insecticide DDT. Biochem. Biophys. Res. Com-
mun. 1990, 166, 1228-1236.
Buser, H. R.; Mu¨ller, M. D. Isomer-selective and enantiomer
selective determination of DDT and related compounds
using chiral high-resolution gas chromatography/mass spec-
trometry and chiral high-performance liquid chromatogra-
phy. Anal. Chem. 1995, 67, 2691-2698.
Meulenberg, E. P.; Mulder, W. H.; Stoks, P. G. Immunoassays
for pesticides. Environ. Sci. Technol. 1995, 29, 553-561.
Mukherjee, I.; Gopal, M. Organochlorine pesticide residues in
dairy milk in and around Delhi. J . AOAC Int. 1993, 76, 283-
286.
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.
Raab, G. M. Comparison of a logistic and a mass-action curve
for radioimmunoassay data. Clin. Chem. 1983, 29, 1757-
1761.
Centeno, E. R.; J ohnson, W. J .; Sehon, A. H. Antibodies to two
common pesticides, DDT and malathion. Int. Arch. Allergy
Appl. Immunol. 1970, 37, 1-13.
Chiu, Y. W.; Carlson, R. E.; Marcus, K. L.; Karu, A. E. A
monoclonal immunoassay for the coplanar polychlorinated
biphenyls. Anal. Chem. 1995, 67, 3829-3839.
Furuya, K.; Urasawa, S. Gas-liquid chromatographic demon-
stration of the specificity of rabbit IgG antibody to the
pesticide DDT and its metabolites. Mol. Immunol. 1981, 18,
95-102.
Goodrow, M. H.; Harrison, R. O.; Hammock, B. D. Hapten
synthesis, antibody development, and competitive inhibition
enzyme immunoassay for s-triazine herbicides. J . Agric.
Food Chem. 1990, 38, 990-996.
Guardino, X.; Serra, C.; Obiols, J .; Rosell, M. G.; Berenguer,
M. J .; Lo´pez, F.; Brosa, J . Determination of DDT and related
compounds in blood samples from agricultural workers. J .
Chromatogr. A 1996, 719, 141-147.
Haas, G. J .; Guardia, E. J . Production of antibodies against
insecticide-protein conjugates. Proc. Soc. Exp. Biol. Med.
1968, 129, 546-551.
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.;
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.
Sherry, J . P. Environmental chemistry: the immunoassay
option. Crit. Rev. Anal. Chem. 1992, 23, 217-300.
Van Emon, J . M.; Lo´pez-AÄ vila, V. Immunochemical methods
for environmental analysis. Anal. Chem. 1992, 64, A79-
A88.
Waliszewski, S. M.; Pard´ıo, V. T.; Waliszewski, K. N.; Chantiri,
J . N.; Infanzo´n, R. M. Levels of organochlorine pesticides
in mexican butter. J . AOAC Int. 1996, 79, 784-786.
Received for review May 19, 1997. Revised manuscript
received J une 23, 1997. Accepted J uly 1, 1997.X
Part of this
work was supported by IMPIVA (Generalitat Valenciana,
Spain), Project 89.93/1035.
J F9704219
X Abstract published in Advance ACS Abstracts, Au-
gust 15, 1997.