Synthesis of haptens and conjugates for ELISAs of phytoestrogens. Development of the immunological tests

Article abstract of DOI:10.1021/jf990896v

Seven carboxylic acid haptens of isoflavonoids were synthesized, with the spacer arm on the oxygen atom at the C7 position for one series, with formononetin, daidzein, equol, biochanin A, and genistein, and at the C8 position for a second series, with only formononetin and daidzein. The different haptens were coupled to bovine serum albumin (BSA) and to swine thyroglobulin (Thyr). Polyclonal antibodies were generated against the BSA conjugates. Enzyme-linked immunosorbent assays (ELISAs) were developed based on competition between free phytoestrogens and the Thyr-hapten conjugates for specific antibodies. IC₅₀ values of the standard curves ranged between 0.8 and 20 ng/mL that is, 0.3 and 9.2 pmol/well. The antibodies obtained should be useful for assays in vegetable matter as well as in biological fluids after a separation step. These ELISAs should be valuable also in the food industry to control phytoestrogen concentrations prior to and after processing.

Full text of DOI:10.1021/jf990896v

J. Agric. Food Chem. 2000, 48, 305−311
305
Syn th esis of Ha p ten s a n d Con ju ga tes for ELISAs of P h ytoestr ogen s.
Develop m en t of th e Im m u n ologica l Tests
Catherine Bennetau-Pelissero,*,† Cyril Le Houe´rou,§ Vale´rie Lamothe, Franc¸oise Le Menn,†
Pierre Babin,‡ and Bernard Bennetau*,§
ENITA de Bordeaux, 1 cours du Ge´ne´ral de Gaulle, 33175 Gradignan, France
Seven carboxylic acid haptens of isoflavonoids were synthesized, with the spacer arm on the oxygen
atom at the C7 position for one series, with formononetin, daidzein, equol, biochanin A, and genistein,
and at the C8 position for a second series, with only formononetin and daidzein. The different haptens
were coupled to bovine serum albumin (BSA) and to swine thyroglobulin (Thyr). Polyclonal antibodies
were generated against the BSA conjugates. Enzyme-linked immunosorbent assays (ELISAs) were
developed based on competition between free phytoestrogens and the Thyr-hapten conjugates for
specific antibodies. IC50 values of the standard curves ranged between 0.8 and 20 ng/mL that is, 0.3
and 9.2 pmol/well. The antibodies obtained should be useful for assays in vegetable matter as well
as in biological fluids after a separation step. These ELISAs should be valuable also in the food
industry to control phytoestrogen concentrations prior to and after processing.
Keyw or d s: Phytoestrogens; isoflavonoids; haptens; polyclonal antibodies; ELISA
INTRODUCTION
that phytoestrogens can protect menopausal women
against osteoporosis (Anderson and Garner, 1998). In
vitro, binding to steroid binding protein (hSHBG and
human RFeto-protein) was shown (Martin et al., 1978;
Garreau et al., 1991). More recently, Kuiper et al. (1998)
showed that genistein 4 (Figure 1) bound to the â form
of the mammalian estradiol receptor. Lately, concern
has grown about potential adverse effects of these
compounds in man, considering the very high levels of
phytoestrogens found in infant formulas of milk sub-
stitutes based on soy (Setchell et al., 1998; Knight et
al., 1998). Indeed, the effect previously demonstrated
in rats after in utero (Hilakivi-Clarke et al., 1998;
Golden et al., 1998) or neonatal exposure (Medlock et
al., 1995; Santti et al., 1998) were examined cautiously.
This issue is still under investigation.
New tools for phytoestrogen assays were developed
recently to measure animal and human exposure, which
is likely to vary from one diet culture to another. Most
of them were immunological tests. Radioimmunoassays
(RIAs) were developed on formononetin 1 (Wang et al.,
1994; Hampl et al., 1998), daidzein 2 (Lape`´ık et al.,
1997), and 4 (Lape`´ık et al., 1998) with haptens to which
a spacer arm was attached on the oxygen atom at the
C7 or C4′ position. To develop an ELISA, we tried to
reproduce the previously reported syntheses. However,
instead of the required acids, complex mixtures were
recovered. Moreover, in the previous syntheses of hap-
tens 8 and 11, the physical and spectroscopic data are
not given. As a result of this lack of information and
difficulty in reproducing the reactions, we decided to
undertake a new process to synthesize the required
haptens with the spacer arm on the oxygen atom at the
C7 position and to synthesize new haptens with the
spacer arm at the C8 position.
Phytoestrogens are natural estrogens present in large
amounts (up to 1-4 mg/g; Anderson and Wolf, 1995) in
numerous vegetables, some of which enter animal and
human food (Farnsworth et al., 1975a,b). They were
demonstrated to be responsible for adverse effects on
animal reproduction and development in mammalian
species, such as sheep (Bennetts et al., 1946), and rats
(Medlock et al., 1995; Levy et al., 1995). Indeed, in sheep
grazing subterranean or red clover, fertility was found
to decrease (Bennetts et al., 1946). A thorough examina-
tion of the endocrine reproductive cycle showed endo-
crine disruptions such as progesterone level impair-
ments, lower rates of embryo implantation (Obst and
Seamark, 1970), or reduced surge of the gonadotrophin
hormone (Findlay et al., 1973; Hughes et al., 1991a,b).
The animals generally exhibited persistent estrous and
increased uterine weight (Braden et al., 1967).
In man, because of lower exposure, only beneficial
effects have been documented so far. Most of the
epidemiological studies compared Western and Asian
consumers. They showed that vegetarian and Asian
consumers, exposed to higher phytoestrogen concentra-
tions, presented lower incidence of estrogen-dependent
diseases such as breast or uterine cancer (Tham, 1998).
More recently soy extracts were shown to decrease the
incidence of menopausal women’s hot flushes (Murkies
et al., 1995). Animal studies also led to the conclusion
* Authors to whom correspondence should be addressed (e-
mail c-bennetau@enitab.fr or b.bennetau@lcoo.u-bordeaux.fr).
† Present address: Laboratoire de Biologie de la Reproduc-
tion des Poissons, Universite´ Bordeaux I, Avenue des Faculte´s,
33405 Talence, France.
§ Present address: LCOO, UMR 5802 CNRS, Universite´
Bordeaux I, 351 cours de la Libe´ration, 33405 Talence, France.
‡ Present address: Laboratoire de Pharmacie Chimique,
Universite´ Bordeaux II, Place de la Victoire, 33000 Bordeaux,
France.
In this paper we report the synthesis of haptens 8-11
of isoflavones 1, 2, biochanin A 3, 4, and equol 5.
Because RIAs use radioisotopes, which are not conve-
nient for control assays in the food industry, we also
10.1021/jf990896v CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/03/2000

306 J. Agric. Food Chem., Vol. 48, No. 2, 2000
Bennetau-Pelissero et al.
8.8 Hz, J ) 2.4 Hz), 7.57 (AA′BB′, 2H), 8.13 (d, 1H, J ) 8.8
Hz), 8.22 (s, 1H); δC (62.9 MHz, acetone-d6) 14.7, 55.8, 62.2,
66.3, 102.5, 114.6, 115.7, 117.3, 125.4, 125.6, 128.5, 131.3,
153.9, 158.8, 160.8, 163.5, 168.9, 206.1.
7-Ethoxycarbonylmethoxy-5-hydroxy-3-(4-methoxyphenyl)-
4H-chromen-4-one (7). A solution of 3 (3 g, 10.5 mmol), ethyl
bromoacetate (3 g, 17.9 mmol), and powdered potassium
carbonate (2.9 g, 21 mmol) in acetone (50 mL) was refluxed
for 48 h (the reaction was monitored by TLC). After cooling,
removal of acetone under reduced pressure gave a crude
product, which was filtered off and purified by washing with
water (50 mL), NaOH (1 M, 30 mL), and water again (30 mL)
to give the product 7 as a white powder (3.7 g, 95%): mp 158
°C; IR (KBr) 3469, 1745, 1656, 1610, 1249, 1182; δH (250 MHz,
DMSO-d6) 1.23 (t, 3H, J ) 7.1 Hz), 3.79 (s, 3H), 4.20 (q, 2H, J
) 7.1 Hz), 4.96 (s, 2H), 6.45 (d, 1H, J ) 2.3 Hz), 6.71 (d, 1H,
J ) 2.3 Hz), 7.02 (AA′BB′, 2H), 7.52 (AA′BB′, 2H), 8.47 (s, 1H),
12.94 (s, 1H); δC (62.9 MHz, DMSO-d6) 13.9, 55.1, 60.8, 65.0,
93.1, 98.4, 105.8, 113.7, 122.2, 122.7, 130.1, 154.8, 157.3, 159.2,
161.7, 163.5, 168.2, 206.7.
7-Carboxymethoxy-3-(4-methoxyphenyl)-4H-chromen-4-one (8).
A solution of sodium carbonate (5% aqueous, 8 mL, excess)
was added to a solution of ester 6 (1 g, 2.82 mmol) in acetone
(50 mL). The mixture was refluxed for 48 h (the reaction was
monitored by TLC). After cooling, removal of the solvent under
reduced pressure, and acidification with HCl (1 M) gave a
crude product, which was filtered off and purified by washing
with water (50 mL) to give the product 8 as a white powder
(800 mg, 87%): mp 214 °C; IR (KBr) 3448, 1736, 1624, 1598,
1249; δH (250 MHz, DMSO-d6) 3.79 (s, 3H), 4.89 (s, 2H), 7.00
(AA′BB′, 2H), 7.09 (d, 1H, J ) 2.3 Hz), 7.14 (dd, 1H, J ) 8.8
Hz, J ) 2.3 Hz), 7.52 (AA′BB′, 2H), 8.04 (s, 1H, J ) 8.8 Hz),
8.42 (s, 1H); δC (50.3 MHz, DMSO-d6) 55.2, 65.2, 101.6, 113.7,
114.9, 118.0, 123.4, 124.1, 127.1, 130.1, 153.6, 157.3, 159.1,
162.2, 169.6, 174.7.
7-Ca r boxym eth oxy-5-h yd r oxy-3-(4-m eth oxyph en yl)-4H -
chromen-4-one (9). To a solution of ester 7 (850 mg, 2.29 mmol)
in acetone (25 mL) was added sodium carbonate (5% aqueous,
10 mL, excess). The mixture was refluxed for 48 h (the reaction
was monitored by TLC). After cooling, removal of acetone
under reduced pressure and acidification with HCl (1 M) gave
a crude product, which was filtered off and purified by washing
with water (50 mL) to give the product 9 as a white powder
(710 mg, 91%): mp > 250 °C; IR (KBr) 3426, 1766, 1736, 1648,
1613, 1249, 1179; δH (250 MHz, DMSO-d6) 3.71 (s, 3H), 4.74
(s, 2H), 6.33 (d, 1H, J ) 2.2 Hz), 6.57 (d, 1H, J ) 2.3 Hz), 6.93
(AA′BB′, 2H), 7.43 (AA′BB′, 2H), 8.37 (s, 1H), 12.85 (s, 1H),
13.2 (s,1H); δC (62.9 MHz, DMSO-d6) 55.1, 65.1, 93.1, 98.4,
105.6, 113.7, 122.1, 122.7, 130.1, 154.7, 157.3, 159.1, 161.6,
163.7, 169.4, 180.2.
7-Carboxymethoxy-3-(4-hydroxyphenyl)-4H-chromen-4-one
(10). BBr3 (1 M in dichloromethane, 54 mL, 53.68 mmol) was
added dropwise with a syringe to a stirred solution of ester 6
(3.17 g, 8.95 mmol) in dichloromethane (30 mL). After complete
addition, the mixture was stirred for 24 h at room temperature
and then was poured onto icy water (500 mL). Filtration of
the crude product gave a yellow residue, which was purified
by chromatography on a column of silica gel eluted with 50%
diethyl ether in dichloromethane and then with 20% ethanol
in dichloromethane to give the product 10 as a yellow powder
(2 g, 71%): mp > 250 °C; IR (KBr) 3265, 1741, 1716, 1616,
1599, 1252; δH (250 MHz, DMSO-d6) 4.90 (s, 2H), 6.83 (AA′BB′,
2H), 7.11 (dd, 1H, J ) 8.8 Hz, J ) 2.4 Hz), 7.15 (d, 1H, J )
2.4 Hz), 7.41 (AA′BB′, 2H), 8.04 (d, 1H, J ) 8.8 Hz), 8.38 (s,
1H), 9.57 (s, 1H), 12.4 (s, 1H); δC (50.3 MHz, DMSO-d6) 65.0,
101.4, 114.7, 114.9, 126.9, 130.0, 153.2, 117.9, 122.3, 123.6,
157.1, 157.2, 162.0, 169.5, 174.6; MS, m/z (relative intensity)
312 (100 M+), 254 (11.9 M+ - CH2CO2), 195 (46.5), 137 (15.8),
118 (40.4). C17H12O6 required: C, 65.39%; H, 3.87%; O, 30.74%.
Found: C, 65.85%; H, 3.89%; O, 28.79%.
F igu r e 1. Structures of isoflavonoids.
present the synthesis of hapten-protein conjugates, the
production of the corresponding antibodies, and the
ELISA tests. The cross-reactivity of the antibodies will
be also discussed.
EXPERIMENTAL PROCEDURES
Ch em ica ls. Organic starting materials for the hapten
synthesis were obtained from Sigma-Aldrich Chimie s.a.r.l. (St.
Quentin Fallavier, France), Acros Organics France (Noisy,
France), and Lancaster Synthesis Ltd. (Bishheim, France).
Thin-layer chromatography (TLC) was performed on 0.25 mm
precoated silica gel 60 F254 plastic sheets from SDS (Peypin,
France). Column chromatography was carried out on silica gel
(SDS 200, 60 Å CC, 35-70 µm, Peypin, France). Diethyl ether
and dichloromethane were dried over 4 Å molecular sieves
(SDS, Peypin France) and then distilled from calcium hydride.
Ethanol was dried by treatment with magnesium followed by
heating under reflux and distillation. Dimethylformamide and
pyridine were distilled from barium oxide. Acetone was dried
over 4 Å molecular sieves and then distilled under nitrogen.
All reagents for biological work were purchased from Sigma-
Aldrich Chimie s.a.r.l. except for the second antibody, that is,
swine immunoglobulin anti-rabbit immunoglobulin coupled to
horseradish peroxidase purchased from Dako (Trappes, France).
In str u m en ts. Melting points were determined on a Kofler
apparatus or Mettler FP 62 capillary melting point apparatus
and are uncorrected. Infrared spectra were recorded on a
Paragon 1000 Perkin-Elmer with polystyrene as standard;
wavenumber (cm-1) values are given. Proton magnetic reso-
nance spectra (1H NMR) were recorded on a Bruker AC 200
at 200 MHz or on a Bruker AC 250 spectrometer at 250 MHz.
Carbon magnetic resonance spectra (13C NMR) were recorded
on a Bruker AC 250 spectrometer at 62.9 MHz or on a Bruker
AC 200 at 50.3 MHz. Chemical shifts (δ) are given in parts
per million (ppm) using tetramethylsilane as the internal
standard at 0.00 ppm. Electron impact mass spectra were
determined on a VG Auto Spec-Q apparatus at 70 eV; data
are reported as m/z (relative intensity). The ELISA technique
was carried out on microtitration plates (NUNC maxisorp)
with 96 wells. The optical densities (OD) were read on a Dynex
MRX II microtitration plate reader at 490 nm. Positive controls
were wells receiving coating and specific antibody without any
free phytoestrogen. The standard curves were expressed
according to the formula ODsample/ODpositivecontrol ) f[log(concen-
trations)].
Syn th eses of Ha p ten s. The syntheses of haptens 8-12 are
described in Figure 2, and the syntheses of haptens 20 and
21 are shown in Figure 3. The structures of the compounds
were verified by spectral methods.
7-Ethoxycarbonylmethoxy-3-(4-methoxyphenyl)-4H-chromen-
4-one (6). A solution of 1 (3 g, 11.2 mmol), ethyl bromoacetate
(3.8 g, 22.7 mmol), and powdered potassium carbonate (3 g,
22 mmol) in acetone (50 mL) was refluxed for 48 h (the reaction
was monitored by TLC). After cooling, removal of the solvent
under reduced pressure gave a crude product, which was
filtered off and purified by washing with water (50 mL), NaOH
(1 M, 30 mL), and water again (30 mL) to give the product 6
as a white powder (3.2 g, 81%): mp 145 °C; IR (KBr) 1747,
1637, 1605, 1252; δH (250 MHz, acetone-d6) 1.27 (t, 3H, J )
7.1 Hz), 3.83 (s, 3H), 4.25 (q, 2H, J ) 7.1 Hz), 4.94 (s, 2H),
6.98 (AA′BB′, 2H), 7.06 (d, 1H, J ) 2.4 Hz), 7.12 (dd, 1H, J )
7-Ca r boxym eth oxy-5-h yd r oxy-3-(4-h yd r oxyph en yl)-4H -
chromen-4-one (11). BBr3 (1 M in dichloromethane, 38 mL, 37.8
mmol) was added dropwise with a syringe to a stirred solution
of ester 7 (2 g, 5.4 mmol) in dichloromethane (40 mL). After
complete addition, the mixture was stirred for 24 h at room

ELISAs for Phytoestrogens
J. Agric. Food Chem., Vol. 48, No. 2, 2000 307
F igu r e 2. Syntheses of haptens 8-12.
palladium (10% on charcoal, 2 g, 5 mol %) was stirred
overnight under hydrogen atmosphere. Because of highly
inflammable properties, hydrogen was manipulated according
to the classic safety instructions. The mixture was filtered and
the filtrate evaporated to give a white solid residue, which was
purified by chromatography on a column of silica gel, eluted
with 40% methanol in dichloromethane, to give the product
12 as a white powder (350 mg, 47%): mp 217 °C; IR (KBr)
3333, 1742, 1613, 1240, 1164; δH (250 MHz, acetone-d6) 2.78-
2.94 (m, 2H), 3.03-3.11 (m, 1H), 3.91-4.00 (m, 1H), 4.16-
4.24 (m, 1H), 4.66 (s, 2H), 6.37 (d, 1H, J ) 2.6 Hz), 6.48 (dd,
1H, J ) 8.4 Hz, J ) 2.6 Hz), 6.82 (AA′BB′, 2H), 7.00 (d, 1H, J
) 8.4 Hz), 7.16 (AA′BB′, 2H), 9.32 (s, 1H), 13.0 (s, 1H); δC (62.9
MHz, DMSO-d6) 31.1, 36.9, 64.5, 70.3, 101.7, 107.1, 114.8,
115.2, 128.3, 130.1, 131.4, 154.5, 156.1, 157.0, 170.2.
1-(2,4-Dihydroxy-3-methylphenyl)-2-(4-methoxyphenyl)eth-
anone (15). BF3‚Et2O (200 mL, 1.61 mol) was added dropwise
to a mixture of 2-methylresorcinol 13 (10 g, 80.55 mmol) and
benzylic acid 14 (13.4 g, 80.63 mmol). The mixture was heated
for 12 h at 70 °C. After cooling, the mixture was poured onto
icy water. The product was filtered off and recrystallized from
chloroform to give the deoxybenzoin 15 as beige crystals (15.3
g, 70%): mp 176-178 °C; IR (KBr) 3284, 1622, 1609, 1584,
1514, 1496, 1359, 1327, 1312, 1247, 1219, 1104, 1031; δH (250
MHz, acetone-d6) 2.05 (s, 3H), 3.75 (s, 3H), 4.19 (s, 2H), 6.50
(d, 1H, J ) 8.8 Hz), 6.87 (AA′BB′, 2H), 7.25 (AA′BB′, 2H), 7.46
(d, 1H, J ) 8.8 Hz), 9.42 (s, 1H), 13.11 (s, 1H); δC (62.9 MHz,
acetone-d6) 7.7, 44.2, 55.5, 107.9, 114.7, 130.9, 131.2, 112.0,
113.1, 128.2, 159.5, 163.1, 164.5, 203.9.
7-Hydroxy-3-(4-methoxyphenyl)-8-methyl-4H-chromen-4-
one (16). BF3‚Et2O (45 mL, 224.76 mmol) was added dropwise
to a stirred solution of deoxybenzoin 15 (15.3 g, 56.2 mmol) in
dimethylformamide (DMF) (150 mL) at room temperature. The
mixture was heated to 50 °C, and mesyl chloride (15 mL,
168.57 mmol) was added with a syringe. The solution was
warmed to 70 °C and stirred for 3 h. After cooling, the mixture
was poured onto icy water (700 mL). Filtration of the crude
product gave a yellow residue, which was recrystallized from
chloroform to give the isoflavone 16 as a white powder (11 g,
70%): mp 292 °C; IR (KBr) 3166, 1620, 1593, 1575, 1510, 1417,
1291, 1268, 1245, 1176; δH (250 MHz, DMSO-d6) 2.23 (s, 3H),
3.78 (s, 3H), 6.99 (AA′BB′, 2H), 7.01 (d, 1H, J ) 8.7 Hz), 7.52
(AA′BB′, 2H), 7.83 (d, 1H, J ) 8.7 Hz), 8.40 (s, 1H), 9.67 (s,
1H); δC (62.9 MHz, DMSO-d6) 7.9, 55.1, 110.8, 113.5, 113.7,
116.6, 122.7, 123.7, 124.3, 130.0, 153.1, 155.4, 158.8, 159.9,
174.9.
F igu r e 3. Syntheses of haptens 20 and 21.
temperature and then was poured onto icy water (400 mL).
Filtration of the crude product gave a yellow residue, which
was purified by chromatography on a column of silica gel
eluted with 50% diethyl ether in dichloromethane and then
with 20% ethanol in dichloromethane to give the product 11
as a yellow powder (2 g, 71%): mp > 250 °C; IR (KBr) 3105,
1721, 1660, 1611, 1250, 1177; δH (250 MHz, DMSO-d6) 4.70
(s, 2H), 6.37 (d, 1H, J ) 1.8 Hz), 6.58 (d, 1H, J ) 1.8 Hz), 6.82
(AA′BB′, 2H), 7.38 (AA′BB′, 2H), 8.38 (s, 1H), 9.43 (s, 1H),
12.92 (s, 1H), 13.1 (s, 1H); δC (62.9 MHz, DMSO-d6) 66.6, 92.9,
98.5, 105.2, 115.0, 120.9, 122.4, 130.0, 157.3, 157.5, 161.5,
164.4, 169.7, 180.3; MS, m/z (relative intensity) 328 (100 M+),
270 (7.3 M+ - CH2CO2), 211 (21.2), 153 (8.6), 118 (21.7).
C
17H12O7 required: C, 62.20%; H, 3.68%; O, 34.12%. Found:
7-Acetoxy-3-(4-methoxyphenyl)-8-methyl-4H-chromen-4-one
(17). Acetic anhydride (25 g, 244.9 mmol) was added dropwise
to a stirred solution of isoflavone 16 (5 g, 17.71 mmol) in
pyridine (80 mL). The mixture was heated to 120 °C for 24 h.
C, 62.57%; H, 3.78%; O, 33.47%.
7-Ca rboxymethoxy-3,4-dihydro-3-(4-hydroxyphenyl)-2H-
chromene (12). A mixture of acid 10 (765 mg, 2.45 mmol) and

308 J. Agric. Food Chem., Vol. 48, No. 2, 2000
Bennetau-Pelissero et al.
Ta ble 1. Titer Eva lu a ted fr om OD a t 490 n m w ith 2.5
After cooling and removal of the solvent under reduced
pressure, a brown solid residue was obtained and then purified
by column chromatography, eluting with 10% ethyl acetate
in dichloromethane to give the product 17 as a white powder
(5.1 g, 89%): mp 168 °C; IR (KBr) 1755, 1636, 1607, 1584,
1511, 1430, 1369, 1293, 1248, 1219, 1208, 1187, 1179; δH (250
MHz, DMSO-d6) 2.27 (s, 3H), 2.38 (s, 3H), 3.79 (s, 3H), 7.01
(AA′BB′, 2H), 7.29 (d, 1H, J ) 8.7 Hz), 7.53 (AA′BB′, 2H), 8.02
(d, 1H, J ) 8.7 Hz), 8.56 (s, 1H); δC (62.9 MHz, DMSO-d6) 8.8,
20.5, 55.1, 113.6, 120.1, 123.6, 130.0, 154.0, 119.7, 121.6, 123.3,
123.7, 152.4, 154.6, 159.0, 168.5, 175.0.
8-Bromomethyl-7-acetoxy-3-(4-methoxyphenyl)-4H-chromen-
4-one (18). A well-stirred mixture of acetate 17 (9 g, 27.75
mmol), N-bromosuccinimide (5 g, 28.05 mmol), and benzoyl
peroxide (0.5 g) in dry carbon tetrachloride (100 mL) was
refluxed for 4 h. After cooling, the mixture was filtered, and
the filtrate was evaporated to give a yellow solid residue, which
was purified by column chromatography, eluting with 5%
diethyl ether in dichloromethane to give the product 18 as a
white powder (10.6 g, 70%): mp 182 °C; IR (KBr) 1763, 1642,
1610, 1513, 1432, 1174, 1142, 1026; δH (250 MHz, DMSO-d6)
2.03 (s, 3H), 3.77 (s, 3H), 5.25 (s, 2H), 6.96 (AA′BB′, 2H), 7.09
(d, 1H, J ) 8.9 Hz), 7.50 (AA′BB′, 2H), 8.00 (d, 1H, J ) 8.9
Hz), 8.37 (s, 1H); δC (62.9 MHz, DMSO-d6) 20.6, 29.7, 55.1,
108.8, 113.5, 114.4, 116.5, 123.1, 124.0, 127.4, 130.0, 152.9,
156.0, 158.9, 161.3, 170.4, 174.6.
7-Acetoxy-8-[2,2-bis(ethoxycarbonyl)ethyl]-3-(4-methoxyphen-
yl)-4H-chromen-4-one (19). A solution of bromide 18 (6 g, 14.88
mmol), diethyl malonate (8 g, 50 mmol), and powdered
potassium carbonate (6.6 g, 47.7 mmol) in DMF (60 mL) was
stirred at room temperature for 4 days (the reaction was
monitored by TLC). The mixture was diluted with water and
dichloromethane (100 mL and 100 mL) and acidified (pH 1-2)
with HCl (1 M). The organic layer was washed with water (2
× 50 mL) and dried over magnesium sulfate. Removal of the
solvent gave a yellow solid residue, which was used to obtain
hapten 20. A small fraction was purified by column chroma-
tography, eluting with 5% diethyl ether in dichloromethane
to give 19 as a yellow solid (10 g): mp 122-124 °C; IR (NaCl)
1731, 1649, 1609, 1584, 1514, 1432, 1370, 1251, 1176, 1037;
δH (200 MHz, acetone-d6) 1.40 (t, 6H, J ) 7.2 Hz), 2.62 (s, 3H),
3.68 (d, 2H, J ) 7.6 Hz), 4.07 (s, 3H), 4.08 (t, 1H, J ) 7.6 Hz),
4.37 (m, 4H), 7.23 (AA′BB′, 2H), 7.53 (d, 1H, J ) 8.8 Hz), 7.83
(AA′BB′, 2H), 8.38 (d, 1H, J ) 8.8 Hz), 8.59 (s, 1H); δC (62.9
MHz, acetone-d6) 14.3, 21.0, 23.7, 51.6, 55.6, 62.1, 114.5, 121.3,
125.2, 131.0, 153.9, 120.8, 123.1, 125.0, 125.2, 156.8, 160.7,
169.2, 169.3, 175.9.
8-(2-Ca r boxyeth yl)-7-h yd r oxy-3-(4-m eth oxyph en yl)-4H -
chromen-4-one (20). To a solution of 19 (10 g) in acetone (70
mL) was added sodium carbonate (5% aqueous, 100 mL,
excess). The mixture was refluxed for 24 h (the reaction was
monitored by TLC). After cooling, removal of acetone under
reduced pressure and acidification with HCl (1 M) gave a crude
product, which was filtered off and purified by washing with
dichloromethane (50 mL) to give the hapten 20 as a white
powder (2,7 g, 53% from 18): mp > 250 °C; IR (KBr) 3327,
1702, 1621, 1611, 1578, 1512, 1439, 1268, 1254, 1239, 1178,
1044; δH (250 MHz, DMSO-d6) 2.45 (m, 2H), 3.02 (m, 2H), 3.78
(s, 3H), 6.98 (AA′BB′, 2H), 7.02 (d, 1H, J ) 8.8 Hz), 7.53
(AA′BB′, 2H), 7.86 (d, 1H, J ) 8.8 Hz), 8.40 (s, 1H), 10.79 (s,
1H), 12.2 (s, 1H); δC (62.9 MHz, DMSO-d6) 18.3, 32.8, 55.1,
113.6, 113.8, 114.1; 116.7, 122.7, 124.3, 124.5, 130.0, 153.1,
155.3, 158.9, 160.0, 173.9, 174.9.
µg/m L Coa tin g a n d 1/4000 An tibod y Dilu tion a
antiserum
hapten
titer
P 01234
P 49636
P 14422
P 37159
P 01114
8
9
10
11
12
+++
+
+++
-
++
P 49639
P 49550
20
21
+++
+++
a
Low titer, -, OD < 1; moderate titer, +, 1 e OD < 1.5; high
titer, ++, 1.5 e OD < 2; extremely high titer, +++, 2 e OD <
2.5.
(d, 1H, J ) 8.8 Hz), 8.39 (s, 1H), 9.57 (s, 1H), 10.78 (s, 1H),
12.2 (s, 1H); δC (62.9 MHz, DMSO-d6) 18.3, 32.8, 113.8, 114.1,
114.9, 116.7, 122.6, 123.0, 124.5, 130.0, 152.8, 155.3, 157.1,
159.9, 173.8, 175.0; MS, m/z (relative intensity) 308 (100 M+
- H2O), 266 (11.6 M+ - H2O - CH2CO), 191 (11.7), 149 (17.8),
118 (34.7). C18H14O6 required: C, 66.26%; H, 4.32%; O, 29.42%.
Found: C, 65.64%; H, 4.59%; O, 28.59%.
P r ep a r a tion of Con ju ga tes. Haptens 8-12, 20, and 21
were coupled either to BSA (for injection to rabbits) or to Thyr
(for coating). The conjugation procedure was adapted from
those of Riggles et al. (1990) and Szurdoki et al. (1992). Briefly,
0.072 mmol of tributylamine (17.3 µL), 0.036 mmol of isobutyl
chloroformate (4.7 µL), and 0.036 mmol of hapten were
dissolved in 1 mL of DMF at 4 °C and then stirred on an ice
bath for 30 min. The resulting activated hapten solution in
DMF was added dropwise to the protein solution (BSA, 0.72
µmol, 6.1 mg; Thyr, 0.072 µmol, 48.24 mg) in 5 mL of borate
buffer (0.2 M borate-boric buffer, pH 8.7). The mixture was
stirred for 6 h at room temperature and then dialyzed, against
phosphate-buffered saline (PBS; 0.01 M, pH 7.4, 0.9% NaCl)
and then against distilled water. The hapten-protein conju-
gates were lyophilized and stored at -20 °C.
Im m u n ization of th e Rabbits. Immunization was achieved
on New Zealand rabbits from CEGAV (France). Rabbits were
first ear-sampled for pre-immunserum test. Hapten-BSA
conjugates (500 µg) were injected each time. For the first
injection the conjugate was dissolved in 1 mL of PBS-complete
Freund’s adjuvant (v/v). For subsequent injections the antigen
was dissolved in PBS-incomplete Freund’s adjuvant (v/v).
Injections were performed at multiple points according to the
following injection schedule. The first three injections were
performed at 1-week intervals. Two additional injections
followed thereafter at a 3-week interval. One week after the
fifth injection, a test was performed on a 5 mL blood sample
to check the specificities and titers of antibodies. Two final
injections were then performed at a 1-month interval. Fifty
milliliter blood samples were collected 1 week after the last
injection. Serum was obtained after blood clotting at 4 °C, for
24 h, and centrifuged at 3000g for 10 min at 4 °C. Sera were
stored at -20 °C in small aliquots. The efficiency of the
immunization was tested in ELISA (as described below) by a
direct binding of the antibody onto the coated conjugate. Titers
of all antisera were compared using the same Thyr-conjugate
coating antigens (2.5 µg/mL) and comparing the OD for a
1/4000 antibody dilution (see Table 2).
Assa y P r oced u r e. Coating of the wells was performed with
the Thyr-hapten conjugates (200 µL/well) in solution in
carbonate buffer (0.05 M, pH 9.6) at 4 °C, overnight. The same
hapten was used for coating as for immunization; that is, the
assays were hapten homologous. Concentrations of conjugates
are listed in Table 3. The wells were then saturated with PBS-
T-PS-DMSO (PBS containing 0.1% porcine serum, 0.05%
Tween 20, and 1% DMSO) at 37 °C for 30 min. Plates were
rinsed three times with PBS-T-DMSO (PBS, 0.05% Tween
20, 1% DMSO). Serial dilutions of the analyte in PBS-T-PS-
DMSO were prepared as standard curves, and 100 µL/well was
added to the plate (see Table 3). Specific antibodies (see
concentrations in Table 3 in PBS-T-PS-DMSO) were then
added (100 µL/well). The incubation lasted for 2 h at 37 °C.
The plates were washed three times with PBS-T-DMSO.
Then 200 µL/well of the second antibody was added in PBS-
8-(2-Ca r boxyeth yl)-7-h yd r oxy-3-(4-h yd r oxyph en yl)-4H -
chromen-4-one (21). BBr3 (1 M in dichloromethane, 22 mL, 21.5
mmol) was added dropwise with a syringe to a stirred solution
of hapten 20 (1.22 g, 3.58 mmol) in dichloromethane (30 mL).
After complete addition, the mixture was stirred for 24 h at
room temperature and then was poured onto icy water (300
mL). Filtration of the crude product gave a yellow residue,
which was recrystallized from ethanol/water (1:1, v/v) to give
the product 21 as a yellow powder (875 mg, 75%): mp > 300
°C; IR (KBr) 3243, 1702, 1623, 1514, 1437, 1293, 1265, 1213;
δH (250 MHz, DMSO-d6) 2.51 (m, 2H), 3.05 (m, 2H), 6.85
(AA′BB′, 2H), 7.04 (d, 1H, J ) 8.8 Hz), 7.42 (AA′BB′, 2H), 7.90

ELISAs for Phytoestrogens
J. Agric. Food Chem., Vol. 48, No. 2, 2000 309
Ta ble 2. Op tim a l Con d ition s for Ea ch Assa y P r oced u r e
antiserum
P 01234
P 14422
P 01114
P 49636
P 37159
P 49639
P 49550
coating (µg/mL)
0.1
0.08
0.5
0.15
2.5
0.1
0.5
specific antibody dilution
competitor
1/30000
1
1/50000
2
1/12000
5
1/50000
3
1/2000
1/60000
1
1/110000
2
4
standard curve limit (ng/mL)
250-0.122
62.5-0.03
0.8
0.51 ( 0.08
1/1000
250-0.122
250-0.122
250-0.122
500-0.24
7.8
0.73 ( 0.09
1/1000
1000-0.48
a
IC50
5
10
20
5
15.6
slope
0.54 ( 0.09
1/2000
0.55 ( 0.08
1/1000
0.35 ( 0.11
1/1000
0.49 ( 0.17
1/2000
0.59 ( 0.06
1/1000
second antibody dilution
a
IC50, concentration of the analyte required for 50% inhibition of the antibody binding to the coating antigen.
Ta ble 3. Sp ecificity of th e ELISAs tow a r d Selected P h ytoestr ogen sa
antiserum
1
2
5
3
4
P 01234 antiserum - compound 1
P 14422 antiserum - compound 2
P 01114 antiserum - compound 5
P 49636 antiserum - compound 3
P 37159 antiserum - compound 4
1.75 ( 0.25
0.013 ( 0.005
0.26 ( 0.15
51 ( 10
6.1 ( 3.0
0.012 ( 0.001
45 ( 10
0.015 ( 0.004
0.8 ( 0.2
12 ( 8
0.013 ( 0.005
2.0 ( 0.5
0.12 ( 0.03
0.75 ( 0.12
40 ( 15
0.025 ( 0.005
0.04 ( 0.002
0.054 ( 0.006
0.60 ( 0.15
20 ( 5
P 49639 antiserum - compound 1
P 49550 antiserum - compound 2
23.6 ( 0.32
0.026 ( 0.002
0.12 ( 0.05
26.6 ( 2.8
15.9 ( 6.5
3.9 ( 0.7
41.3 ( 6.0
50 ( 8
a
Results are expressed as percent cross-reactivity values: (IC50 of parent compound/IC50 of compound) × 100 ((SD calculated on
three tests).
T-PS-DMSO (see Table 3 for concentrations). The incubation
was performed at 37 °C for 30 min. To measure peroxidase
activity, 200 µL/well of substrate solution containing 0.005 M
o-phenylenediamine (10 mg in 20 mL) and 0.00025% H2O2 30%
(5 µL in 20 mL) in citrate-phosphate buffer (0.15 M, pH 5.0)
was added. The reaction took place at room temperature for
30 min and was stopped with 50 µL/well of 4 M H2SO4. OD
values were read at 490 nm. The standard curves counted 12
points in duplicates with a 2-fold increase between concentra-
tions.
Cr oss-Rea ctivity Tests. The cross-reactivities of the an-
tibodies were tested in the competitive way, according to the
optimal conditions summarized in Table 3. In that case besides
a standard curve obtained with the analyte, the antibody was
exposed to serial dilutions of other phytoestrogens in PBS-
T-PS-DMSO. The concentrations of the competitors varied
from 50 to 0.003 µg/mL with a 4-fold decrease between
concentrations. Results are given in Table 3.
yields by selective demethylation with boron tribromide
(Bhatt and Kulkarni, 1983). Hydrogenation of 10 af-
forded the hapten 12, suitable for the production of
antibodies against equo 5.
C8 Hapten Syntheses. To improve the specificity of
antibodies, we tried to avoid a direct conjugation using
a hydroxyl group of isoflavoneS, which is an important
antigenic determinant of this class of compounds. By a
variation of the Wa¨ha¨la¨ method (Wa¨ha¨la¨ and Hase,
1991), we obtained the methylated deoxybenzoin 15
(Figure 3). After protection of hydroxyl groups with
acetic anhydride, the brominated compound 18 was
obtained by bromination with N-bromosuccinimide and
a catalytic amount of benzoyl peroxide in carbon tetra-
chloride (Zammattio et al., 1991). A malonic ester
synthesis [see, for example, Szurdoki et al. (1992) and
cited references] afforded the hapten 20. Treatment of
the latter with boron tribromide, in the same conditions
as above, gave the hapten 21.
RESULTS AND DISCUSSION
Syn th etic Wor k . Five carboxylic acid haptens of
isoflavonoids were synthesized with the spacer arm at
the O7 position (8-12) and two haptens (20 and 21)
with the spacer arm at the C8 position.
Con ju ga tion Tests. The conjugations obtained, ac-
cording to the method described above, were tested for
their efficiency following the spectrophotometric method
described by Erlanger et al. (1957).
Hapten Series with Spacer Arm on the Oxygen Atom
at C7. As mentioned earlier, the attempted reactions,
following previous procedures, to synthesize the re-
quired acids did not provide the expected haptens and,
instead, complex mixtures were recovered. Moreover, a
previous report on hapten syntheses by regioselective
alkylation of genistein at either the 4′-O or 7-O position
did not present detailed experimental procedures (La-
pe`´ık et al., 1998, and references cited therein). Surpris-
ingly, the cited reference (Wa¨ha¨la¨ et al., 1995), in the
experimental procedure for chemicals, deals only with
the synthesis and labeling of isoflavones. Therefore, we
turned our attention to devising a new process to
synthesize the required acids.
Im m u n iza tion Tests. According to Table 1, the titers
of the antibodies were extremely high except that of P
37159. In that case we injected four different rabbits
(not listed here) with the same results.
ELISA Tests. The optimal conditions of the ELISAs
are shown in Table 2. The standard sigmoid curves
obtained for each of the antibodies raised are presented
in Figures 4 and 5. In the series functionalized on the
oxygen atom at the C7 position, the sensitivities of the
assays are generally good with standard curve limits
between 250 and 0.244 ng/mL and IC50 around 10 ng/
mL. In that respect P 14422 is somehow different
because its standard curve limits are 62.5 and 0.0304
ng/mL and its IC50 is at 0.8 ng/mL. This could be related
to the conjugation ratio obtained with BSA, which is
the best of the series. The slopes obtained in this series
were all rather similar (Figure 4). However, in some
cases, the variability calculated on nine different assays
is high, possibly due to an adsorption phenomenon on
glassware or microtitration plates. As a matter of fact,
we used silicone-coated glassware, silicone-coated tips,
and buffers with 1% DMSO to reduce the adsorption
phenomenon.
Starting from 1 and 3 (Figure 2), obtained as previ-
ously reported (Pelissero et al., 1991), the spacer arm
was bonded to the oxygen atom at the C7 position using
ethyl bromoacetate with potassium carbonate in ac-
etone. Saponification of esters 6 and 7 afforded the
corresponding acids 8 and 9. With 3, the C7 hydroxyl
2
group was alkylated selectively according to D NMR
data. This could be explained by the presence of a
hydrogen bond between the hydroxyl group and the B
ring carbonyl. Haptens 10 and 11 were obtained in good

310 J. Agric. Food Chem., Vol. 48, No. 2, 2000
Bennetau-Pelissero et al.
et al. (1994) reported for 1 an IC50 of 0.4 ng/mL.
However, previous data indicated that phytoestrogens
were present in high concentrations in food (Anderson
and Wolf, 1995) as well as in animal and human fluids
(Setchell, 1985). Moreover, phytoestrogens were con-
sidered to be 1000-10000-fold less estrogenic than
estradiol (Setchell, 1985). Thus, a very high sensitivity
assay is not required for quantification in food or
measurements of physiological relevance.
Cr oss-Rea ctivity of th e An tibod ies. The results
obtained on cross-reactivities are presented in Table 3.
It can be seen that cross-reactions of all phytoestrogens
with P 14422 are low. Inversely, the other antisera
exhibit very low cross-reactions with 5. This can be
interpreted in terms of the steric and electronic struc-
tural features of 5, which are very different from those
of isoflavones. On the other hand, high levels of cross-
reactions are recorded between other compounds from
the two series. In the group of haptens functionalized
on the oxygen atom at the C7 position, the highest cross-
reactions are registered between 2 and 4; P 37159
recognizes 2 at 45%, and P 14422 recognizes 4 at 40%.
This means that the additional hydroxyl group in 11
on the C5 position is not recognized by the antibodies.
The same phenomenon is observed with P 49636, which
recognizes 1, at 51%. Wang et al. (1994) obtained the
same results with their antibody anti-formononetin 7-O-
carboxymethyl ether.
F igu r e 4. Standard curves obtained with the different
antibodies in the O7 series. Points are the mean of duplicates.
Analyte-antibody combinations are listed in Table 2.
In the group of haptens functionalized at the C8
position, the specificity is worse. High levels of cross-
reactions are observed between P 49550, 1, and 4,
whereas P 49639 strongly recognizes 2 and 3. In that
case the cross-reaction between 2 and 1 could be
expected with respect to the similarity between OMe
and OH groups at C4′ (Sherry, 1992). However, this
cross-reactivity was higher in the C8 series than in the
O7 series. As a matter of fact, in the first series, the
short spacer arm at position 7 projected the opposite
moiety (position 4′) of the hapten molecule for strong
recognition in the immunizing conjugate. Thus, the
spacer arm directed the specificity of the O7-derived
antibody series toward the immunodominant part (posi-
tion 4′) of the hapten (Szurdoki et al., 1995) In the C8
series, the position of the linker at the hapten appears
to allow more conformational freedom of the hapten in
the immunizing conjugates; thus, the selectivity of the
resulting antisera is lower. Lape`´ık et al. (1994, 1998)
worked with the molecules substituted in 4′ and on the
oxygen atom at C7. As we showed in the C8 series, they
obtained high cross-reactions between 1 and 2 as well
as between 3 and 4.
In conclusion, the antibodies obtained are valuable
for assays of phytoestrogens (Picherit et al., unpublished
results). However, because of some high cross-reactions,
separation of the compounds, prior to assay, should be
worthwhile. In addition, because of high phytoestrogen
levels in foods and because of low biological activities
of these compounds, the sensitivity of our assays ap-
pears to be sufficient. Moreover, ELISAs are easier to
use than RIAs because they do not need radioisotopes.
All of these facts make our ELISAs very easy and
suitable for measurements of phytoestrogen levels of
physiological relevance in animal fluids and for diet
manufacturers to control their materials before and
after processing.
F igu r e 5. Standard curves obtained with the different
antibodies in the C8 series. Points are the mean of duplicates.
Analyte-antibody combinations are listed in Table 2.
In the C8 series, the sensitivities are lower. For P
49550 the standard curve limits are 1000 and 0.488 ng/
mL and the IC50 is at 15.6 ng/mL, and for P 49639 the
standard curve limits are 500 and 0.244 ng/mL with an
IC50 at 7.8 ng/mL. This could be due to a nonspecific
recognition of the Thyr-hapten conjugates. Our sensi-
tivity results are of the same order as those obtained
with ELISA of two herbicides (norflurazon and bro-
macil), described by Riggles et al. (1990) and Szurdoki
et al. (1992). Indeed, they obtained IC50 values between
1 and 10 ng/mL, that is, 1-10 µg/L or 1-10 ppb, as we
did. When we compared our ELISAs to RIAs of phy-
toestrogens, we observed that the latter are generally
10-100 times more sensitive than the former. Lape`´ık
et al. (1997) obtained for 2 an IC50 of 0.170 ng/mL and
for 4 an IC50 of 0.311 or 0.150 ng/mL depending on the
antibody used (Lape`´ık et al., 1998). In addition, Wang

ELISAs for Phytoestrogens
J. Agric. Food Chem., Vol. 48, No. 2, 2000 311
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BSA, bovine serum albumin; DMF, N,N-dimethylfor-
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linked immunosorbent assay; IC50, inhibiting concen-
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