Competitive Inhibition ELISA for Thiram
J. Agric. Food Chem., Vol. 48, No. 10, 2000 4493
(methyl)amino]butanoate 3 (0.78 g, 3.3 mmol), and sodium
dimethyldithiocarbamate (1.17 g, 8 mmol) in 20 mL of methanol/
dichloromethane (15:5) was stirred for 20 h at room temper-
ature and concentrated in vacuo. The residue was dissolved
into water (20 mL), acidified (pH 3) with 3 M HCl, and
extracted with dichloromethane (2 × 15 mL). The organic
phase was dried with MgSO4, filtered, and concentrated in
vacuo. The product, 1, (Rf: 0.2) was purified by flash chroma-
tography (ethyl acetate/hexane, 1:2). Yield: 0.3 g (27%) amor-
vidual dithiocarbamates or their metabolites has been
the aim of many workers. For example a specific high
performance liquid chromatography (HPLC) method for
determination of thiram, salts of alkylenebis(dithiocar-
bamic acids), and N,N′-dimethyl(dithiocarbamic acid)
has been reported by Gustafsson and Thompson (1981).
Approaches to specific determination of thiram (Irth et
al., 1986, 1990; Baumann et al., 1991) and EBDCs
(Miles and Zhou, 1991; Irth et al., 1990) have been
examined in a number of laboratories. Validation of
HPLC method in which the EBDCs can be separated
from thiram has been reported by J ongen et al. (1991).
More recently Lo et al. (1996) have developed an HPLC
and atomic absorption method to distinguish propineb,
zineb, maneb, and mancozeb fungicides. However none
of the above methods can be performed directly on
foodstuff. All of them need an initial extraction step and
require sophisticated equipment such as HPLC or gas
chromatography (GC), available in only well-equipped
centralized laboratories. They are more labor-intensive,
time-consuming, and expensive than the reference
method. Furthermore the complete identification of
residues of the complex DTCs still remains impossible.
The problem of monitoring the residual level of
pesticides in foodstuffs need the development of simple,
rapid, and sensitive procedures adequate for handling
a large number of samples. In this way, immunoassay
has been proved to fulfill such analytical requirement,
provided that appropriate antibodies are available
(Kaufman and Clower, 1995; Ferguson et al., 1993).
Immunoassay may offer either an alternative to the
traditional method or the potential for an initial rapid
screening method to reduce the number of samples
requiring chemical analysis. So immunoassays have
been developed for some pesticides. In the case of
dithiocarbamates, there is no report of an immunoassay
development.
1
phous powder (mp: 105-106 °C, dec). H NMR (400.13 MHz,
CDCl3): δ 2.0 (p, 2H, J ) 7 Hz), 2.45 (t, 2H, J ) 7 Hz), 3.75,
4.1 (2t, 2H, J ) 7 Hz), 3.32, 3.36, 3.50, 3.55 (4s, 9H), 5.33 (s,
2H,S-CH2-S), 10.7 (s, 1H). 13C NMR (100.62 MHz, CDCl3):
δ 21.3, 22.0 (C-CH2-C), 30.5, 30.8 (CH2-CO2H), 39.6, 43.6
(N-CH3), 41.5, 45.5 ((CH3)2N-), 46.2 (S-CH2-S), 53.3, 55.8
(N-CH2-), 178.1, 178.6 (C)O), 195.0, 195.8 ((CH3)N-C)S),
195.13 ((CH3)2N-C)S). Anal. Calcd for C10H18N2O2S4: C,
36.78; H, 5.56. Found: C, 36.62; H, 5.51. Two symmetrical
products were also obtained (Rf ) 0.9, C7H14N2S4; Rf )0.05,
C13H22N2O4S4).
{[(Dialkylamino)carbothioyl]sulfanyl}alkanoic Acid 2. Gen-
eral Procedure. To a water solution (25 mL) of sodium
hydroxide (1.2 g, 30 mmol) and 40% aqueous solution of
dimethylamine (2.25 g, 20 mmol) was added dropwise, at room
temperature, carbon disulfide (1.9 g, 25 mmol). After 30 min,
a mixture of 6-bromohexanoic acid (3.9 g, 25 mmol), sodium
hydroxide (0.8 g, 20 mmol), and water (15 mL) was added. The
mixture was stirred for 20 h, acidified (pH 3) with 3 M HCl,
and extracted using dichloromethane (2 × 20 mL). The organic
phase was dried with MgSO4, filtered, and concentrated in
vacuo. The crude product was dissolved into dichloromethane
and precipitated by hexane at 0 °C to give 2c (3.2 g, 13.6 mmol,
68% yield), a white powder (mp: 85-86 °C, dec).
1
The structure of haptens 2 was fully ascertained using H
and 13C NMR spectroscopy and by comparison with reported
data.
2-{[(Dimethylamino)carbothioyl]sulfanyl}ethanoic Acid 2a .
(Garraway, 1962). White powder (mp: 145-146 °C, dec). 1H
NMR (99.54 MHz, CDCl3): δ 3.4, 3.5 (2s, 6H), 4.15 (s, 2H),
11.2 (s, 1H). 13C NMR (25.05 MHz, CD3OD/CDCl3): δ 39.2
(CH2), 41.4, 45.5 (N(CH3)2), 170.5 (C)O), 195.2 (C)S).
4-{[(Dimethylamino)carbothioyl]sulfanyl}butanoic Acid 2b.
(Garraway, 1962). White powder (mp: 92-93 °C, dec). 1H NMR
(99.54 MHz, CDCl3): δ 2.05 (p, 2H, J ) 7 Hz), 2.5 (t, 2H, J )
7 Hz, CH2-CO2H), 3.35 (t, 2H, J ) 7 Hz), 3.4 (s, 3H), 3.55 (s,
3H). 13C NMR (25.05 MHz, CDCl3): δ 23.9 (CH2), 32.9 (CH2-
CO2H), 36.3 (CH2-S), 41.4, 45.2 (N(CH3)2), 178.8 (C)O), 196.5
(C)S).
In the present paper, we describe the development of
an enzyme-linked immunosorbent assay (ELISA) for the
detection of thiram using anti-thiram polyclonal anti-
bodies. In this aim, different haptens, belonging to two
families, were synthesized and used in order to obtain
antibodies. They were also screened for their capacity
to improve ELISA sensitivity in heterologous competi-
tive ELISA.
6-{[(Dimethylamino)carbothioyl]sulfanyl}hexanoic Acid 2c.
(Garraway, 1962). White powder (mp: 85-86 °C, dec). 1H NMR
(99.54 MHz, CDCl3): δ 1.2-1.9 (m, 6H), 2.35 (t, 2H, J ) 7 Hz,
CH2-CO2H), 3.25 (t, 2H, J ) 7 Hz, S-CH2-), 3.32 (s, 3H),
3.50 (s, 3H). 13C NMR (25.05 MHz, CDCl3): δ 24.2, 28.3, 28.4,
33.9 (CH2-CO2H), 37.2 (S-CH2-), 180 (C)O), 197.3 (C)S).
11-{[(Dimethylamino)carbothioyl]sulfanyl}undecanoic Acid
MATERIALS AND METHODS
Bovine serum albumin (BSA), ovalbumin (Ova), and goat
anti-rabbit IgG were obtained from SIGMA. Other chemicals
were from Aldrich. Thin-layer chromatography and column
chromatography were carried out using Merck silica gel 60.
1
2d . White powder (mp: 90-92 °C, dec). H NMR (99.54 MHz,
CDCl3): δ 1.1-2 (m, 16H), 2.4 (t, 2H, J ) 7 Hz), 3.3 (t, 2H, J
) 7 Hz), 3.5 (s, 6H), 10.2 (s, 1H). 13C NMR (25.05 MHz,
CDCl3): δ 24.6, 28.5, 28.9 (2C), 29.1 (2C), 29.3, 32.8, 34.1
(CH2-CO2H), 37.6 (CH2-S), 41.4, 45.3 (2 -CH3), 180.4 (C)O),
197.5 (C)S).
1
The structure of haptens was confirmed by H and 13C NMR
spectroscopy (Brucker DRX 400 or J EOL FX 100) using
tetramethylsilane as internal standard.
Ha p ten Syn th esis. Disodium 4-[Carbodithioato(methyl)-
amino]butanoate 3. To a water solution (50 mL) of sodium
hydroxide (1.2 g, 30 mmol) and 4-(methyl-amino)butanoic acid
chlohydrate salt (1.53 g, 10 mmol) was added dropwise, at
room temperature, carbon disulfide (0.91 g, 12 mmol). After 1
h, the mixture was warmed to 40 °C for 2 h. Then, the mixture
was extracted with acetone (2 × 20 mL), and the acetone phase
was discarded. The product, 3, precipitated as a white powder.
2-{[(Diethylamino)carbothioyl]sulfanyl}ethanoic Acid 2e.
White powder (mp: 88-90 °C, dec). 1H NMR (99.54 MHz,
CDCl3): δ 1.35 (t, 6H, J ) 7 Hz), 3.95 (m, 4H), 4.26 (s, 2H),
11.9 (s, 1H). 13C NMR (25.05 MHz, CDCl3): δ 11.1, 12.2 (2
-CH3), 38.3 (CH2-CO2H), 46.9, 49.9 (2 -CH2), 173.8 (C)O),
192.8 (C)S).
P r ep a r a tion a n d Ch a r a cter iza tion of Ha p ten -P r otein
Con ju ga tes. All haptens used in this study contained a free
carboxylic group suitable to react with amine groups of
proteins. Hapten-protein conjugations were carried out by the
N-hydroxysuccinimide (NHS) active ester method according
to Langone and Van Vunakis (1975).
1
Yield: 2 g (84%). H NMR (99.54 MHz, D2O): δ 1.8-2.05 (m,
2H), 2.2 (t, 2H, J ) 7 Hz), 3.45 (s, 3H), 4.05 (t, 2H, J ) 7 Hz).
13C NMR (25.05 MHz, CDCl3): δ 25.4 (C-CH2-C), 36.5 (CH2-
CO2-), 44.8 (N-CH3), 58.1 (CH2-N), 184.2 (C)O), 209, 0
(C)S).
5,11-Dimethyl-6,10-dithioxo-7,9-dithia-5,11-diazadodecano-
ic Acid, Hapten 1. A mixture of disodium 4-[carbodithioato-
The carboxylic acid hapten (50 mg) was dissolved in 1.0 mL
of dimethylformamide (DMF) with 70 mg of NHS and 60 mg