Development of an enzyme-linked immunosorbent assay for bisphenol A using chicken immunoglobulins

Article abstract of DOI:10.1021/jf0202739

Bisphenol A was coupled, after derivatization into a suitable hapten, to bovine serum albumin and ovalbumin in order to produce immunizing and coating antigens. The immunizing antigens were injected into chickens, which allowed the isolation of specific bisphenol A immunoglobulins from the egg yolk. These antibodies were used in an indirect competitive enzyme-linked immunosorbent assay for the determination of bisphenol A in aqueous solutions. Various parameters, influencing the assay sensitivity, were evaluated. The applicability of the assay for the determination of bisphenol A in milk was also studied. The assay was not as sensitive as other analytical techniques used in bisphenol A analysis, since typical I₅₀ levels of 2.5 μM were reached in aqueous solutions. This study nevertheless illustrates the usefulness and the potency of chicken antibodies in the analysis of migration residues from packaging materials using immunochemical techniques. In addition, the assay showed to be quite specific for bisphenol A as well. Only for bisphenol A analogues, cross reactivities of about 40% were reached, enabling the use of the antibodies for the screening of bisphenol A and alike compounds.

Full text of DOI:10.1021/jf0202739

J. Agric. Food Chem. 2002, 50, 5273−5282 5273
Development of an Enzyme-Linked Immunosorbent Assay for
Bisphenol A Using Chicken Immunoglobulins
BRUNO DE MEULENAER,* KATLEEN BAERT, HEIKKI LANCKRIET,
VERA VAN HOED, AND ANDRE HUYGHEBAERT
Laboratory of Food Chemistry and Analysis, Department of Food Technology and Nutrition, Ghent
University, Coupure Links 653, B-9000 Ghent, Belgium
Bisphenol A was coupled, after derivatization into a suitable hapten, to bovine serum albumin and
ovalbumin in order to produce immunizing and coating antigens. The immunizing antigens were
injected into chickens, which allowed the isolation of specific bisphenol A immunoglobulins from the
egg yolk. These antibodies were used in an indirect competitive enzyme-linked immunosorbent assay
for the determination of bisphenol A in aqueous solutions. Various parameters, influencing the assay
sensitivity, were evaluated. The applicability of the assay for the determination of bisphenol A in milk
was also studied. The assay was not as sensitive as other analytical techniques used in bisphenol
A analysis, since typical I50 levels of 2.5 µM were reached in aqueous solutions. This study
nevertheless illustrates the usefulness and the potency of chicken antibodies in the analysis of
migration residues from packaging materials using immunochemical techniques. In addition, the assay
showed to be quite specific for bisphenol A as well. Only for bisphenol A analogues, cross reactivities
of about 40% were reached, enabling the use of the antibodies for the screening of bisphenol A and
alike compounds.
KEYWORDS: Bisphenol A; immunoassay; IgY; ELISA; migration; chicken immunoglobulins; water; milk;
cross reactivity; xeno-estrogens
INTRODUCTION
of the development of the male sexual organs has been studied
in several animals such as mice, rats (26), and fish (27). Negative
effects on sperm quality due to exposure to bisphenol A have
been reported as well (28, 29).
Bisphenol A (2,2-bis(4-hydroxyphenyl)propane) is an im-
portant monomer for the production of high quality food contact
materials such as epoxy coatings (1) and polycarbonate (2).
Because of incomplete polymerization or partial hydrolysis of
the polymer, bisphenol A migration from a packaging material
to the food occurs as revealed by several reports (1, 3-9). Apart
from being a food contaminant, it should be stressed that
bisphenol A is considered an environmental contaminant as well,
which, for example, is reported to be present in the sediment
of rivers (10, 11).
Because of the available toxicity data, it can be concluded
that dietary exposure to bisphenol A is a food safety problem.
Apart from an acute toxicity (12), the xeno-estrogenic effects,
already known for a number of decades (13), are an important
issue. Recent research revealed that the estrogenic activity is a
multicause phenomenon. First of all, it has been proved that
bisphenol A binds to the so-called estrogen receptors (14-17).
The substance also enhances cell proliferation in general (18-
20) and the cell proliferation of the female sexual organs in
particular (21-24). Furthermore, bisphenol A is reported to
induce prolactine secretion (23, 25). Therefore, the influence
of bisphenol A on all stages of development and in particular
In addition to the xeno-estrogenic effects, the possible
carcinogenicity of bsiphenol A was studied as well. Although
bisphenol A is not a considered a mutagen (30) and although
reports linking bisphenol A exposure to cancer incidence are
limited (31, 32), a number of observations illustrate that indeed
bisphenol A exposure should be considered with some concern
in this respect. The substance is reported to affect mitosis (33,
34) and to induce aneuploidy (34-36). In addtion, bisphenol
A metabolites have been shown to react with DNA inducing
the production of DNA adducts (35-38). As already mentioned,
bisphenol A may induce cell proliferation as well.
Because of its toxicity, the analysis of bisphenol A residues
in foods has received some attention during the last 5 years as
illustrated above. In these studies, classic instrumental analytical
techniques have been used such as gas chromatography and
high-performance liquid chromatography (HPLC) most fre-
quently combined with mass spectrometric detection and
extensive sample clean up techniques as reviewed by De
Meulenaer and Huyghebaert (39). Recently, however, the use
of immunochemical analytical techniques has been reported for
bisphenol A analysis. Both monoclonal (40) and polyclonal (41)
mammalian antibodies were used successfully in immunosorbent
* To whom correspondence should be addressed. Tel: ++ 32 9 264 61
66. Fax: ++ 32 9 264 62 18. E-mail: Bruno.Demeulenaer@rug.ac.be.
10.1021/jf0202739 CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/17/2002

5274 J. Agric. Food Chem., Vol. 50, No. 19, 2002
De Meulenaer et al.
used as a chromogen, the substrate buffer (pH 5.0) was a 40 mM citric
acid and 35 mM Na2HPO4‚12H2O solution. The substrate solution
consisted of 40 mg of OPD in 100 mL of substrate buffer to which
just before use 5 mL of 0.03% (v/v) H2O2 was added. The stop solution
was 2.5 M HCl. If ABTS was used as a chromogen, the substrate buffer
(pH 4.0) consisted of 0.05 M tri-sodium citrate in water. The substrate
solution consisted of 30 mg of ABTS in 100 mL of substrate buffer to
which just before use 5 mL of 6% (v/v) H2O2 was added. The stop
solution was a 0.1 M NaF, 0.008 M NaOH, and 0.001 M Na2EDTA
solution.
assays. The monoclonal antibodies were selected in such a way
that a high resistance toward organic solvents was achieved,
enabling the detection of bisphenol up to the 1 ppb range in
solutions containing up to 50% of methanol (40). The polyclonal
antibodies enabled the detection of bisphenol A in the 0.5-5
ppb range in urine samples (41). Although the use of immuno-
chemical techniques in the analysis of food contaminants is well-
established, the present paper and those mentioned above are
the first to consider them for the analysis of migration residues
from food contact materials. It should be stressed as well that
immunochemical techniques are particularly useful for screening
purposes. Such screening techniques are presently, however, not
described for bisphenol A, which considering its possible
presence in both food and in the environment, is a considerable
disadvantage.
Aqueous solutions of bisphenol A and the substances for which cross
reactivity was evaluated were prepared as follows. One gram of
substance was dissolved in 50 mL of methanol, and this solution was
diluted in water up to the desired concentration. Methanol concentration
in the final solutions was considered to be negligible (constant
concentration 1.5% v/v). For aqueous methanolic bisphenol A solutions,
in which higher concentrations of methanol were used, methanol was
added additionally until the desired concentration was reached.
Hapten Synthesis. Synthesis of 4-[1-(4-{[tert-Butyl(dimethyl)silyl]-
oxy}phenyl)methylethyl]phenol 2. To a solution of 2.28 g (10 mmol)
of bisphenol A 1 in 50 mL of DMF, 1.70 g (25 mmol) of imidazole
was added. Subsequently, a solution of 1.51 g (10 mmol) of tBCDS in
20 mL of DMF was added dropwise at RT. The reaction mixture was
stirred for 1 h at RT and poured out in 100 mL of water. The mixture
was extracted with hexane (2 × 50 mL and 2 × 25 mL) thus avoiding
the coextraction of unreacted bisphenol A. The organic phase was dried
over sodium sulfate and evaporated to dryness under reduced pressure.
The white residue was dissolved in 5 mL of hexane-ethyl acetate 6:1
(v/v) and further purified by column chromatography. Therefore, a glass
column (32 mm internal diameter) was filled with 60 g of silica gel
using hexane-ethyl acetate 6:1 (v/v) as a mobile phase. Elution was
accomplished by gravity. From the fraction eluting between 160 and
350 mL, 1.11 g (32%) of pure product could be obtained after
Although chicken immunoglobulins are less frequently used
in immunochemical techniques in comparison with mammalian
antibodies, they offer some distinct advantages as emphasized
in a recent review (42). Therefore, the present paper considered
the use of chicken immunoglobulins in an enzyme immunosor-
bent assay (ELISA) for bisphenol A analysis.
MATERIALS AND METHODS
Reagents and Buffers. 4,4′-Dihydroxybenzophenon 99%, 4,4′-
ethylidenebisphenol 99%, 4-cumylphenol 99%, bis-(4-hydroxyphenyl)-
methane 98%, p-cresol 99%, m-cresol 99%, 4-hydroxydiphenylmethane
99%, 4,4′-cyclohexylidenebisphenol 98%, 2,2-bis-(4-hydroxyphenyl)-
perfluorpropane 97%, bis-(4-hydroxyphenyl)sulfone 98%, 4,4′-(1,4-
phenylene-diisopropylidene)bisphenol 98%, 4,4′-isopropylidene bis(2,6-
dimethylphenol) 98%, 3,4′-isopropylidene-diphenol 98%, 4,4′-(1,3-
phenylenediisopropylidene)bisphenol 99%, 1,4-dihydroxybenzene, 4,4′-
dihydroxybiphenyl 97%, butylbenzyl phthalate, 4-butylphenol, and 4,4′-
(1-phenylethylidene) bisphenol 99% were from Aldrich Chemical Co.,
U.S.A. Benzoic acid, sodium hydrogen carbonate, methanol, hexane,
and sodium hydroxide were obtained from Chem-Lab, Belgium.
Butylhydroxyanisol was from Koch-light laboratories, England. BADGE
was a generous gift from Ciba Specialty Chemicals, Belgium. Anhy-
drous disodium carbonate, 1,3-dihydroxybenzene, sodium chloride,
hydrochloric acid 25%, DMF, THF, diethyl ether, chloroform, tri-
sodium citrate dihydrate and gelatine were purchased from UCB,
Belgium. Phenol 99%, 4-nonylphenol (mixture of isomers) 99%,
bisphenol A 97%, potassium dihydrogen phosphate, disodium hydrogen
phosphate dodecahydrate, dibutylphthalate, imidazole, tBCDS, anhy-
drous sodium sulfate, N-hydroxy succinimide, 4-(dimethylamino)-
pyridine, glutaric anhydride, citric acid, TBAF, N,N′-dicyclohexylcar-
bodiimide, benzyl alcohol, anhydrous sodium sulfite, sodium tertaborate
decahydrate, sodium fluoride, disodium ethylenediaminetetraacetic acid
(EDTA), and ammonium sulfate were from Acros Organics, U.S.A.
BSA (fraction V, 96%), OVA (Grade III), Freund’s incomplete
adjuvant, Freund’s complete adjuvant, 98% ABTS, 95% TNBS, and
Tween 20 were from Sigma Chemical.
Hydrogen peroxide 30%, OPD, silica gel G60, ethyl acetate, and
potassium chloride were from Merck, Germany. HRP conjugated rabbit
antichicken IgG was from ICN Biomedicals Inc., U.S.A. All of these
reagents were of analytical grade unless otherwise mentioned.
Potassium caseinate and the skimmed milk powder were generous
gifts of Rovita, Germany, and Belgomilk, Belgium, respectively.
Sunflower oil was obtained from Vandemoortele (Belgium). Sephadex
G25 was purchased from Pharmacia (Sweden) and was equilibrated
for at least 16 h in an excess of PBS prior to use. Dried THF was
obtained by continuous reflux of THF over sodium using benzophenon
as an indicator.
PBS (pH 7.4) consisted of 0.135 M NaCl, 1.5 mM KH2PO4, 8 mM
Na2HPO4‚12H2O, and 2.7 mM KCl. The coating buffer (pH 9.6) was
a 15 mM Na2CO3 and a 35 mM NaHCO3 solution. The dilution buffer
(PBS-Tween 20) consisted PBS with 0.05% (v/v) Tween 20. The wash
solution was 0.05% (v/v) Tween 20 solution in 0.15 M NaCl. The
blocking solution was PBS with 3% (w/v) K-caseinate. If OPD was
1
evaporation to dryness under reduced pressure. H NMR (CDCl3): δ
0.08 (s, 6H, SiCH3), 0.86 (s, 9H, tBu), 1.49 (s, 6H, CH3), 6.56 (d, 2H,
aromatic), 6.63 (d, 2H, aromatic), 6.95 (d, 4H, aromatic). 13C NMR
(CDCl3): δ -4.26 (2 × CH3-Si), 18.29 (Cquat , tBu), 25.82 (tBu),
31.20 (2 × CH3), 41.80 (C-CH3), 114.82 (2 × CH, aromatic), 119.42
(2 × CH, aromatic), 127.80 and 128.05 (4 × CH, aromatic), 143.50
and 143.77 (2 × C aromatic), 153.20 and 153.30 (Cquat-OH and Cquat
-
OSi). MS m/z (%): 343 (41); 329 (27); 328 (80); 286 (21); 136 (18);
135 (100); 107 (11); 73 (20). IR (cm-1): νmax 3272 (OH); 1511 (Ph);
1252 (Si(CH3)2). Melting point: 79.5 °C.
Synthesis of 5-{4-[1-(4-{[tert-Butyl(dimethyl)silyl]oxy}phenyl)-
1-methylethyl]phenoxy}-5-oxopentanoic Acid 3. To a solution of
1368 mg (4 mmol) of 2 in 30 mL of dry THF, 13.68 g (120 mmol) of
glutaric anhydride and 489 mg of 4-(dimethylamino)pyridine (4 mmol)
were added. The mixture was heated to reflux for 2 h, poured out in
50 mL of water, acidified with 10 N HCl to pH < 2, and extracted
with chloroform (2 × 50 mL and 2 × 25 mL). The organic phase was
dried over sodium sulfate and evaporated to dryness under reduced
pressure. The residue was dissolved in 20 mL of diethyl ether and
cooled for 2 h at -18 °C in order to precipitate part of the glutaric
anhydride in excess, which was removed by filtration. After it was
evaporated to dryness under reduced pressure, the residue was dissolved
in 2 mL of hexane-ethyl acetate 4:1 (v/v) and further purified using
column chromatography. Therefore, a glass column (10 mm internal
diameter) was filled with 5 g of silica gel and hexane. Elution was
accomplished with 40 mL of hexane-ethyl acetate (4:1) (v/v). After it
was evaporated to dryness under reduced pressure, the residue was
dissolved in 2 mL of hexane-ethyl acetate 4:1 (v/v) and again purified
using a similar chromatographic setup. From the fraction eluting
between 5 and 55 mL, 967 mg (53%) of pure product could be obtained
after evaporation to dryness under reduced pressure. 1H NMR
(CDCl3): δ 0.08 (s, 6H, SiCH3), 0.86 (s, 9H, tBu), 1.54 (s, 6H, CH3),
1.96 (quint, 2H, CH2CH2COOH), 2.41 (t, 2H, CH2COOH), 2.55 (t,
2H, CH2COOPh), 6.62 (d, 2H, aromatic), 6.85 (d, 2H, aromatic), 6.96
and 7.08 (d, 2 × 2H, aromatic). 13C NMR (CDCl3): δ -4.23 (2 ×
CH3-Si), 18.08 (CH2CH2COOH), 19.65 (Cquat , tBu), 25.59 (tBu), 30.91
(2 × CH3), 32.64 (CH2COOPh), 33.11 (CH2COOH), 42.02 (C-CH3),
119.23 (2 × CH aromatic), 120.62 (2 × CH aromatic), 127.62 and

ELISA for Bisphenol A Using Chicken Immunoglobulins
J. Agric. Food Chem., Vol. 50, No. 19, 2002 5275
dilution method described by Akita and Nakai (46). Briefly, V mL of
egg yolk was separated from the egg and diluted with 8 × V mL of
water and pH was set with 1 N HCl between 5.0 and 5.2. After they
were incubated for 16 h at 4 °C and centrifuged (10 000g, 1 h, 4 °C),
the supernatant was filtered. After 72 g of ammonium sulfate and about
170 mL of water was added in order to achieve a 60% saturated solution
of ammonium sulfate, the mixture was incubated for 1 h at RT and
centrifuged (10 000g, 20 min, RT). The residue was dissolved in a 19%
(w/v) sodium sulfate solution. After it was incubated for 20 min at RT
and centrifuged (2000g, 20 min, RT), the residue was dissolved in a
14% (w/v) sodium sulfate solution. After it was incubated for 20 min
at RT and centrifuged (2000g, 20 min, RT), the residue was dissolved
in V/6 mL of PBS and stored in small aliquots at -18 °C. For further
use, they were diluted from this final solution.
Immunoassays. General Conditions. Ninety-six well F 96 Maxisorp
Nunc immunoplates from Nunc (Denmark) were coated with coating
antigen A solution (12.5 µg/mL coating buffer, 100 µL/well) by
overnight incubation at 4 °C in the dark. Plates were washed three
times (200 µL wash solution/well) and blocked (200 µL blocking
solution/well) for 2 h at RT in the dark. Afterward, the plates were
washed twice as previously. To study the immune response of the
chickens or to obtain competition curves, primary antibodies were added
as described elsewhere. Afterward, the plates were washed as described
above (three times).
For the detection reaction, the HRP-conjugated secondary antibody
was added (100 µL/well, 3.4 µ/mL dilution buffer). After it was
incubated for 1 h at 37 °C and the plates were washed (three times),
100 µL/well of substrate solution was added followed by an additional
incubation at 37 °C for 1 h. Finally, 25 µL/well of the appropriate stop
solution was added before measuring the absorbance at the appropriate
wavelength (for OPD 492 nm; for ABTS 405 nm). Absorbances were
corrected for blank readings obtained by using immunoglobulins
isolated from the eggs of nonimmunized chickens. These conditions
were followed unless otherwise stated.
Table 1. Characteristics of Immunizing and Coating Bisphenol A
Antigens
bisphenol A/
protein ratio
during synthesis
(mol/mol)
actual bisphenol A/
protein ratio
antigen
(mol/mol)
immunizing antigen
coating antigen A
coating antigen B
coating antigen C
138.0
90.0
45.0
22.5
26.0 ± 0.26
4.86 ± 0.10
3.80 ± 0.11
2.95 ± 0.03
127.71 (4 × CH aromatic), 142.88 and 148.17 (2 × C aromatic), 148.57
(Cquat-OSi), 153.34 (Cquat-OCO), 171.43 (COOPh), 178.80 (COOH).
MS m/z (%): 88 (24); 86 (90); 84 (100); 49 (26); 47 (30). IR (cm-1):
ν
max 3416 (OH); 1749 (COOPh); 1704 (COOH); 1510 (Ph); 1226 (Si-
(CH3)2).
Synthesis of 5-{4-[1-(4-Hydroxyphenyl)-1-methylethyl]phenoxy}-
5-oxopentanoic Acid 4. To an ice-cooled solution of 790 mg (2.5
mmol) of TBAF in 15 mL of THF, an ice-cooled solution of 1140 mg
(2.5 mmol) of 3 in 15 mL of THF was added dropwise. After 1 h at 0
°C, the reaction mixture was poured out in 30 mL of water, acidifed
with 10 N HCl to pH < 2, and extracted with chloroform (3 × 20
mL). The combined organic fractions were washed with 25% HCl (10
× 10 mL), dried over sodium sulfate, and evaporated to dryness under
1
reduced pressure, yielding 633 mg of pure hapten 4 (74%). H NMR
(CDCl3): δ 1.63 (s, 6H, CH3), 2.06 (quint, 2H, CH2CH2COOH), 2.49
(t, 2H, CH2COOH), 2.67 (t, 2H, CH2COOPh), 6.75 (d, 2H, aromatic),
6.95 (d, 2H, aromatic), 7.07 (d, 2H, aromatic), 7.22 (d, 2H, aromatic).13C
NMR (CDCl3): δ 20.36 (CH2CH2COOH), 31.58 (C-CH3), 33.43 (CH2-
COOPh), 34.08 (CH2COOH), 42.65 (C-CH3), 115.39 (2 × CH
aromatic), 121.33 (2 × CH aromatic), 128.46 and 128.52 (CH aromatic),
143.36 (C aromatic), 149.00 (C aromatic), 149.33 (Cquat-OH), 154.22
(Cquat-OCO), 172.65 (COOPh), 179.43 (COOH). MS m/z (%): 228 (37);
213 (100); 86 (24); 84 (35). IR (cm-1): νmax 3416 (OH); 1749 (COOPh);
1704 (COOH); 1510 (pH); 1226 (Si(CH3)2).
Preparation of Immunizing and Coating Conjugates. Haptens
were covalently attached to BSA and OVA, respectively, using the
method originally described by Cuatrecasas and Parikh (43). For the
synthesis of the immunizing conjugates, 102 mg of hapten 4 (0.3 mmol)
in 3 mL of DMF was mixed with 48 mg (0.4 mmol) of N-hydroxy
succinimide and 62 mg (0.3 mmol) of N,N′-dicyclohexylcarbodiimide.
The solution was left for 16 h at RT, and the crystals formed were
removed by decantation of the supernatant, which was added dropwise
to 10 mL of a protein solution (15 mg BSA/mL in 50 mM sodium
carbonate at pH 9.6). This solution was stirred for 4 h at RT, and finally,
the conjugates were purified by gel filtration on Sephadex G25 using
PBS as mobile phase.
Three different coating conjugates were produced using OVA, with
a varying bisphenol A to protein ratio as summarized in Table 1. Similar
reaction conditions as for the production of BSA-bisphenol conjugates
were used except for the volume of protein solution, since the
concentration in the carbonate buffer was kept constant at 15 mg OVA/
mL.
The extent of coupling for each conjugate was determined using
the TNBS method as described by Fields (44). Keeping into account
the number of available amino groups in BSA and OVA as reported
by Habeeb (45), the bisphenol A load of the immunizing and coating
antigen was estimated as summarized in Table 1.
Indirect ELISA. To study the immune response of the chickens, the
appropriate primary antibody dilution was added to the coated wells
(100 µL/well in dilution buffer) and the plates were incubated for 1 h
at 37 °C. OPD was used as chromogen.
Indirect CompetitiVe ELISA. For the competition step, 50 µL of the
appropriate bisphenol A dilution and 50 µL of the primary antibody
solution were added to each well. Primary antibodies were diluted as
follows: 20 µL of the original primary antibody solution in PBS was
further diluted to 7.24 mL with PBS. Subsequently, 26.64 mL of PBS
containing 0.3% (w/v) BSA, 520 µL of NaOH 0.1 N, and 5.6 mL of
a 4 M NaCl solution were added, obtaining a final dilution of the
primary antibody of 1/2000, a pH of 8.0, a BSA concentration of 0.2%
(w/v), and a calculated ionic strength of 700 mM. This dilution is
referred to as the competition buffer. The plates were incubated for 1
h at 37 °C. Both OPD and ABTS were used as chromogens.
Studies on the Composition of the Competition Buffer. Charac-
teristics of competition buffer were evaluated as follows: ionic strength,
presence of surface active agents, and pH.
For the ionic strength studies, the amount of 4 M NaCl added to the
competition buffer was adjusted in order to vary its ionic strength,
reducing the amount of PBS accordingly, thus keeping the primary
antibody concentration constant for all experiments. If necessary, the
addition of NaCl solution was replaced by the addition of water. The
ionic strength was calculated using the following formula
I ) 0.5 ciz2i
∑
i
Chicken Immunization and Immunoglobulin Isolation. Three Isa
Brown chickens of 40 weeks old were injected intramuscularly with 1
mL of a 1:1 (v/v) mixture of Freund’s complete adjuvant and PBS
containing 500 µg of immunizing antigen (BSA-bisphenol A conju-
gate). After 3 weeks, a supplementary injection of 1 mL of a 1:1 (v/v)
mixture of Freund’s incomplete adjuvant and PBS containing 500 µg
of immunizing antigen was given. Afterward, boaster injections of 500
µg of immunizing conjugate in PBS were repeated every 3 weeks during
a 70 day period, after which the immunization procedure was stopped.
Eggs were collected daily and individually identified. The immuno-
globulins were isolated from the egg yolk using a modified aqueous
where I is the ionic strength, c is the concentration of each ion, and
z is its charge. Ionic strengths reported refer to the ionic strength of
the diluted IgY solution before it is applied in the assay. BSA itself
was not present in the competition buffer for the reported experiment.
The influence of Tween 20 in the competition buffer was evaluated
as a function of the ionic strength and its concentration. Therefore,
BSA was replaced in the competition buffer by Tween 20 (0-0.4%
v/v). In addition to Tween 20, however, BSA (0.2% w/v) and potassium
caseinate (0.2% w/v) were also evaluated.

5276 J. Agric. Food Chem., Vol. 50, No. 19, 2002
De Meulenaer et al.
Figure 1. Bisphenol A hapten synthesis.
The amount of NaOH (0.1 N) or HCl (0.1 N) added to the
competition buffer was adjusted together with the amount NaCl 4 M
and PBS in such a way that the desired pH was reached keeping the
ionic strength constant. Initially, these studies were performed using
Tween 20, but apart from this surfactant, potassium caseinate (0.2%
w/v) and BSA (0.2%) were also used, respectively.
Coating Antigen Studies. Three different coating antigens, as
indicated in Table 1, were used at varying concentrations (0.4-12.5
µg/mL) during the coating of the multiwell plates.
Cross Reactivity. Competitive assays using coating antigen C (0.8
µg/mL in coating buffer) were performed using various structural
bisphenol A analogues in order to determine their respective I50 values
(micromolar). I50 is the concentration of the analyte at which half of
the maximal signal intensity is reached. Cross reactivity was calculated
as
Instruments. The Titertek multiskan plus MK II (U.S.A.) and the
Organon Teknika (The Netherlands) multireaders were used throughout
this research. NMR spectra were obtained using a JEOL PMX 270 SI
(270 MHz) instrument by dissolving the samples in deuterated
chloroform, using tetramethylsilane as a reference. For the mass spectra,
a Varian MAT 112 mass spectrometer (U.S.A.) (70 eV) was used, which
was coupled with a Varian aerograph 2700 gas chromatograph (U.S.A.).
The gas chromatograph was equipped with a CPSil 5CB column
(Chrompack, The Netherlands) (internal diameter 0.32 mm, film
thickness 0.25 µm, length 30 m). A Perkin-Elmer model 1310 infrared
spectrometer was used to obtain IR spectra. For the determination of
the melting point, a Bu¨chi 535 was used.
RESULTS AND DISCUSSION
Bisphenol A Hapten and Antigen Synthesis. To produce
suitable protein conjugates for immunization and coating,
bisphenol A 1 was transformed into hapten 4 according to the
reaction scheme outlined in Figure 1. Therefore, bisphenol A
1 was primarily derivatized with tBCDS to compound 2 in order
to protect one of the hydroxyl groups as described by Corey
and Venkateswarlu (47). Of course, reaction at the second
hydroxyl group of bisphenol A could not be prevented. So,
consequently, compound 2 was isolated from the reaction
mixture by preparative column chromatography. The free
hydroxyl group in compound 2 reacted with an excess of glutaric
anhydride yielding derivative 3 after various purification steps
(selective partial precipitation of glutaric anhydride; column
chromatography). Removal of the protecting group was achieved
using TBAF at low temperatures, similarly as described by Sinha
et al. (48), yielding hapten 4.
This hapten, carrying a five carbon spacer arm, was used for
the production of immunizing and coating antigens. Therefore,
BSA and OVA were used, respectively. Three different coating
antigens were obtained by variation of the bisphenol A to protein
ratio during synthesis as indicated in Table 1.
Chicken Immunization. Response of the chickens toward
the immunization procedure was evaluated by using an indirect
ELISA as described. Two of the immunized chickens reacted
toward the applied immunization procedure. Because one of
cross reactivity (%) ) (I50,bisphenol A)/(I50,compound) × 100
Solvent Effect. Competitive immunoassays using coating antigen
C (0.8 µg/mL in coating buffer) and ABTS as a chromogen were
accomplished using aqueous methanol bisphenol A solutions at the
appropriate methanol and bisphenol A concentrations.
Assay Performance in Milk. Competitive immunoassays using
coating antigen C (0.8 µg/mL in coating buffer) and ABTS as a
chromogen were accomplished using milk samples that were spiked
with bisphenol A at the appropriate concentration. Reconstituted milk
was prepared as follows: 10 g of skimmed milk powder was dissolved
in 60 mL of distilled water. For the addition of bisphenol A, the
appropriate aqueous solution was added at this stage as well. If
necessary, sunflower oil at the appropriate concentration was emulsified
in the dispersion using a Ultraturrax mixer at moderate speed.
Afterward, the mixture was diluted until a final volume of 100 mL
was reached. Pasteurized milk samples at various fat contents were
obtained from retail shops. These samples were spiked with a
concentrated methanolic bisphenol A solution, keeping the methanol
concentration constant at 1.5% v/v.
Data Processing. Competition curves were obtained in 4-fold. When
required, curves were normalized by expressing the experimental
absorbance levels (B) as (B/B0), where B0 is the absorbance in absence
of analyte. Absolute or normalized signals were fitted to a four
parameter logistic equation using a commercial software package (SPSS
10.0).

ELISA for Bisphenol A Using Chicken Immunoglobulins
J. Agric. Food Chem., Vol. 50, No. 19, 2002 5277
Figure 2. Response of a chicken immunized with bisphenol A−BSA
conjugate at various days after start of the immunization (( ) 7 days; 9
) 28 days; 2 ) 42 days; ) ) 45 days; × ) 70 days. Response was
Figure 3. Competition curves with a varying ionic strength of the
competition buffer (9 ) 200 mM; 2 ) 300 mM; × ) 400 mM; O ) 800
mM; ( ) 3000 mM. Responses were normalized to the highest maximal
absorbance observed, B0,max).
normalized to the maximal absorbance observed, B ).
0
these chickens, however, stopped egg production 35 days after
the start of the immunization procedure, a small amount of
useful eggs could be collected for further use. Therefore, only
the eggs from one single chicken were used throughout further
experiments. As can be seen from Figure 2, appreciable
response was observed about 1 month after the immunization
procedure started. In addition, this response could be maintained
until at least 70 days, after which the immunization experiments
were aborted because of the large number of useful eggs already
collected. To the authors knowledge, this is the first reported
successful chicken immunization with plastic monomer-protein
conjugates in general and with bisphenol A-protein conjugates
in particular. Moreover, it should be noted that the use of chicken
antibodies in the analysis of food contaminants is not so well-
documented as compared to the use of other antibodies, despite
the ease of immunization and antibody isolation and purification.
In addition, large quantities of antibodies can be collected
because of the daily egg production and the relatively high
concentration of antibodies in the egg yolk (42).
Figure 4. Influence of Tween 20 concentration at various ionic strengths
Optimization of the Competitive Indirect Bisphenol A
Assay. The isolated antibodies from the chickens were used to
evaluate their use in a competitive indirect ELISA. Therefore,
in the first place, the parameters having an influence on the
assay performance were evaluated. In the preliminary experi-
ments, it was revealed that the composition of the competition
buffer had an important effect on the assay characteristics.
on the I50 (filled symbols) and B (open symbols) values ((,) ) 700
mM; 2,4 ) 1600 mM; 9,0 ) 2000 mM).
0
competitive immunoassays. This was unexpected; therefore,
further attempts to optimize the sensitivity of the assay were
performed.
Influence of Surface ActiVe Compounds. Surface active
compounds such as Tween 20 are frequently applied in
immunoassays in order to reduce nonspecific interactions. As
can be seen from Figure 4, I50 values increased with Tween 20
concentration at several ionic strengths tested. Consequently,
Tween 20 concentration should be as low as possible in order
to achieve better assay sensitivity. If no Tween 20 was present,
however, assay reproducibility was rather poor. Similar observa-
tions were made previously by Abad and Montoya (49). Apart
from the effects on assay sensitivity, Tween 20 affected the
maximum absorbance B0 as well: B0 values became maximal
at relatively low Tween 20 concentrations (0.025% v/v) at all
ionic strengths tested. These observations are not in complete
agreement with those previously reported by Abad and Montoya
(49), who found a constant decrease of the maximal absorbance
as a function of the Tween 20 concentration. For the sake of
completeness, it should be noted that at lower ionic strengths
Ionic Strength Effect. As indicated in Figure 3, ionic strength
of the competition buffer was of prime importance with regard
to the assay performance. Within the range of 400-800 mM,
assay performance was considered to be acceptable within the
achievable sensitivity range. At lower levels, the I50 values were
unacceptably high, while for higher ionic strengths, overall
absorbance became too low. Therefore, an ionic strength of 700
mM was selected as an optimal level. Previously, Abad and
Montoya (49) observed a strong influence of the ionic strength
on the performance of an assay for carbaryl using monoclonal
mice antibodies as well. They suggested that the interaction
between antibodies and hydrophobic analytes is highly influ-
enced by the polarity of the buffers used.
From Figure 3, it was revealed as well that the overall
sensitivity of the assay was rather poor, as compared to other

5278 J. Agric. Food Chem., Vol. 50, No. 19, 2002
De Meulenaer et al.
Other ReleVant Parameters. To obtain good assay charac-
teristics of a competitive ELISA, limiting concentrations of
immunoreagents were required. Therefore, the effect of the
antibody and the HRP-labeled antibody concentration in com-
bination with the incubation time of the competition step and
the detection reaction were evaluated as well. In summary,
lowering the concentration of the primary antibody and the HRP-
labeled antibody resulted both in lower maximal absorbances
and lower I50 values. Similar observations could be made if the
incubation times were restricted. Also, the following blocking
reagents were evaluated for their capability to reduce a-specific
binding: gelatine (0.1-3%, w/v), ovalbumine (3%, w/v),
skimmed milk powder (5%, w/v), and potassium caseinate (3%,
w/v). Milk powder and potassium caseinate proved to be the
best. Potassium caseinate was preferred because of its more
simple chemical composition as compared to skimmed milk
powder.
The use of ABTS as an alternative detecting reagent instead
of OPD was evaluated as well. Maximal absorbance levels
decreased together, however, with the blank signal. In addition,
a significant increase in assay sensitivity was observed as well
(I50 (OPD): 11.36 µM ( 0.67; I50 (ABTS): 2.40 µM ( 0.19).
Figure 5. Influence of the kind of coating antigen and its concentration
on I50 (filled symbols) and B (open symbols) values ((,) ) coating antigen
0
A; 9,0 ) coating antigen B; 2,4 ) coating antigen C; for the
characteristics of these coating antigens, see Table 1).
None of the optimization steps reported changed the assay
sensitivity by orders of magnitude. Therefore, the use of the
assay is currently limited to solutions in which the bisphenol A
concentration is relatively high (hundreds of ppbs). Despite this
fact, the specificity and the applicability of the assay to relevant
samples was evaluated.
than those reported in Figure 4, even higher maximal absor-
bance levels were observed, which was in correspondence with
the data shown in Figure 3.
Apart from Tween 20, caseinate and BSA were also consid-
ered. The use of the caseinate resulted in a slightly improved
assay sensitivity in combination with lower maximal absorbance
levels as compared to Tween 20 (0.1%, w/v). For BSA, no
significant differences as compared to the use of Tween 20
(0.1%, w/v) were observed. This is in contrast to the results
obtained by Abad and Montoya (49), who observed improved
assay characteristics by replacing Tween 20 with BSA.
Specificity of the Assay. Cross reactivity in aqueous solutions
of various structural analogues (phenols) was evaluated. Most
compounds were selected because they can be present in food
contact materials as well, apart from bisphenol A. Some of these
are known to exhibit an estrogenic effect (BADGE, phthalates)
as well (Table 2).
pH Effect. Because bisphenol A can be considered a weak
organic acid, the pH of the competition buffer was tested
together with the use of various surface active agents (Tween
20, caseinate, BSA). Generally, at pH levels lower than five
and higher than ten, very low absorbance levels were obtained
(B0 < 0.2). Within this range, however, the influence of the pH
on the assay performance was not significant for all surfactants
tested (not shown). This was again surprising since pH
dependence of both signal intensity and sensitivity of ELISAs
have been reported (49-51). Remarkably, however, it was
observed that assay reproducibility increased if BSA was used
instead of Tween 20, in a pH-dependent manner (starting from
pH 8 to pH 10). Again, no explanation could be given for these
phenomena.
Compounds with a similar structure as bisphenol A showed
some varied cross reactivity. Removal of central methyl groups
of the bisphenol A molecule resulted in a fairly strong reduction
in cross reactivity, while removal of one of the hydroxyl groups
resulted in a cross reactivity of about 40%. This high cross
reactivity for these particular compounds can probably be
explained by the type of antigen used for immunization, since
one of the hydroxyl groups of the bisphenol A molecule was
used to couple it to the carrier protein. More complex bisphenol
A analogues could not be recognized by the isolated immuno-
globulins. Curiously, simple phenolic compounds such as
4-butylphenol and butylhydroxyanisol showed appreciable cross
reactivity (10%). Phthalates did not show cross reactivity at all,
while BADGE was slightly cross-reactive. This indicates that
there is no link between the estrogenic character of these
compounds and their immunochemical recognition. For the sake
of completeness, it should be mentioned that no significant
variation was observed between the cross reactivity of several
eggs.
Coating Antigens. The influence of the coating antigen on
the assay performance was evaluated as well. In the initial
experiments using coating antigen A, it was observed that lower
antigen concentrations resulted in decreasing I50 values. Un-
fortunately, however, B0 values decreased as well. Because of
these observations, other coating antigens were produced with
a lower bisphenol A load and similar experiments were
performed. A similar trend as in the preliminary experiments
could be observed (Figure 5). It should be noted, however, that
for the antigen with the lowest bisphenol A load tested (coating
antigen C), the decrease in I50 values was less pronounced as
compared to the other antigens. It should also be noted that
although the bisphenol A load of the coating antigen B was
lower as compared to coating antigen A, higher I50 values were
observed at all concentration levels tested. This was surprising
because of the earlier observations.
Influence of Methanol on the Assay. A draft CEN procedure
was prepared for the quantification of bisphenol A in oil. This
method is based on an extraction of bisphenol A from the oil
using an aqueous methanol solution and subsequent HPLC-
UV analysis (52). Therefore, the assay characteristics were
evaluated as a function of the methanol concentration in the
competition buffer. The presence of methanol resulted in a
significant concentration-dependent decrease in maximum signal
and assay sensitivity (Figure 6). At the highest methanol
concentration tested, high assay variability was observed as well.
This is in correspondence with the results obtained by Abad

ELISA for Bisphenol A Using Chicken Immunoglobulins
J. Agric. Food Chem., Vol. 50, No. 19, 2002 5279
Table 2. Cross Reactivity of the Assay

5280 J. Agric. Food Chem., Vol. 50, No. 19, 2002
De Meulenaer et al.
Table 2 (Continued)
Figure 7. Competition curves obtained in skimmed milk at selected ionic
strengths of the competition buffer (aqueous bisphenol A solution: b )
700 mM; bisphenol A in milk: ) ) 140 mM; 0 ) 220 mM; 4 ) 300
mM; 2 ) 400 mM; 9 ) 600 mM).
Figure 6. Influence on methanol during the competition on the I50 (filled
symbols) and B (open symbols).
0
and Montoya (49), who investigated the influence of several
organic solvents on the characteristics of a carbaryl assay. The
presence of 10% (v/v) methanol could be tolerated in order to
apply the assay for the quantification of bisphenol A in oil.
Therefore, the aqueous methanolic extract of the oil could be
applied without significant dilution, illustrating the potency of
the presented assay.
reconstituted milk, however, the reduced assay performance
could not be solely due to an absorption of bisphenol A by the
fat globules rendering it unavailable for immunochemical
reactions (results not shown).
Because milk contains an appreciable amount of salts as well,
the ionic strength of the competition buffer was adjusted because
of the earlier observations with regard to the influence of ionic
strength on the assay performance. By changing the ionic
strength of the competition buffer, the assay performance could
be influenced in such a way that the inhibition curves obtained
in milk were similar to those obtained in water (Figure 7). A
complete match of the competition curves obtained in water
and milk, respectively, is difficult to achieve because of complex
sample matrix. Especially the presence of milk proteins seem
Assay Performance in Milk. Because dairy products are
frequently contacted with polycarbonate, application of the assay
for milk could be of practical interest. Therefore, the perfor-
mance of the assay was evaluated for milk at various fat
contents. Initial experiments on whole reconstituted milk
revealed a strong reduction in assay quality (increased I50 and
reduced B0). Because similar results were obtained for skimmed

ELISA for Bisphenol A Using Chicken Immunoglobulins
J. Agric. Food Chem., Vol. 50, No. 19, 2002 5281
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ABBREVIATIONS USED
ABTS, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid);
BADGE, bisphenol A diglycidyl ether; BSA, bovine serum
albumin; ELISA, enzyme-linked immunosorbent assay; CEN,
Centre Europe´en de Normalization, European Centre for
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yolk immunoglobulins; OPD, orthophenylenediamine; OVA,
ovalbumin; PBS, phosphate-buffered saline; RT, room temper-
ature; THF, tetrahydrofurane; DMF, dimethylformamide; TBAF,
N,N,N-tributyl-1-butylammonium fluoride; tBDCDS, tert-butyl-
(chloro)dimethylsilane; TNBS, 2,4,6-trinitrobenzenesulfonic
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