Development of enantioselective polyclonal antibodies to detect styrene oxide protein adducts

Article abstract of DOI:10.1021/ac8023262

Styrene has been reported to be pneumotoxic and hepatotoxic in humans and animals. Styrene oxide, a major reactive metabolite of styrene, has been found to form covalent binding with proteins, such as albumin and hemoglobin. Styrene oxide has two optical

Full text of DOI:10.1021/ac8023262

Anal. Chem. 2009, 81, 2668–2677
Development of Enantioselective Polyclonal
Antibodies to Detect Styrene Oxide Protein
Adducts
Shuijie Shen,† Fan Zhang,‡ Su Zeng,† Ye Tian,† Xiaojuan Chai,† Shirley Gee,§
Bruce D. Hammock,§ and Jiang Zheng*,‡
Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang
University, Hangzhou, Zhejiang, 310058, China, Center for Developmental Therapeutics, Seattle Children’s Hospital
Research Institute, Division of Gastroenterology, Department of Pediatrics, University of Washington,
Seattle, Washington 98101, and Department of Entomology, University of California, Davis, California 95616
Styrene has been reported to be pneumotoxic and hepa-
totoxic in humans and animals. Styrene oxide, a major
reactive metabolite of styrene, has been found to form
covalent binding with proteins, such as albumin and
hemoglobin. Styrene oxide has two optical isomers and it
was reported that the (R)-enantiomer was more toxic than
the (S)-enantiomer. The purpose of this study was to
develop polyclonal antibodies that can stereoselectively
recognize proteins modified by styrene oxide enantiomers
at cysteine residues. Immunogens were prepared by
alkylation of thiolated keyhole limpet hemocyanin (KLH)
with styrene oxide enantiomers. Polyclonal antibodies
were raised by immunization of rabbits with the chiral
immunogens. Titration tests showed all six rabbits gener-
ated high titers of antisera that recognize (R)- or (S)-
coating antigens accordingly. No cross-reaction was ob-
served toward the carrier protein (BSA). All three rabbits
immunized with (R)-immunogen produced antibodies that
show enantioselectivity to the corresponding antigen,
while only one among the three rabbits immunized with
(S)-immunogen generated antibodies with enantioselec-
tivity of the recognition. The enantioselectivity was also
observed in competitive ELISA and immunoblot analysis.
Additionally, competitive ELISA tests showed that the
immunorecognition required the hydroxyl group of
the haptens. Immunoblot analysis demonstrated that the
immunorecognition depended on the amount of protein
adducts blotted and hapten loading in protein adducts.
In summary, we successfully developed polyclonal anti-
bodies to stereoselectively detect protein adducts modi-
fied by styrene oxide enantiomers.
automobile parts.1 Workplace exposures to styrene are the highest
in factories fabricating reinforced plastics products. Humans are
potentially exposed to this widespread environmental contami-
nant.1-3 Styrene causes both pneumotoxicity and hepatotoxicity
in animals and humans. Styrene is mainly metabolized by
cytochrome P450 to styrene oxide (2, SO), which was reported
to be cytotoxic, mutagenic, and potentially carcinogenic. Styrene
oxide is a known electrophilic species chemically reactive to
nucleophiles of macromolecules, such as nucleotides and proteins,
to form covalent binding adducts both in vitro and in vivo.4-6 It
has been reported that SO can covalently bind to a variety of
nucleophilic functional groups in proteins, including the sulfhydryl
group of cysteine, imidazole of histidine, the carboxylic acid group
of aspartic and glutamic acid, amino group of lysine, and the
N-terminal amino group of proteins.5,7-9 Among those nucleophilic
amino acids, cysteine has been reported as the most reactive
amino acid residue to be modified by SO.8,10 Albumin and
hemoglobin SO adducts have been identified both in humans and
animals. A dose-response relationship between the quantities of
SO adducts and styrene exposure was observed in styrene-
exposed workers.11,12 Formation of protein covalent binding with
xenobiotics or their active metabolites has long been recognized
as a possible mechanism of chemical toxicity.13 Immunochemical
approaches have been successfully developed to detect protein
(1) Miller, R. R.; Newhook, R.; Poole, A. Crit. Rev. Toxicol. 1994, 24
(Supplement), S1–S10
(2) Carrillo-Carrio´n, C.; Lucena, R.; Ca´rdenas, S.; Valca´rcel, M. J. Chromatogr.,
A 2007, 1171, 1–7
(3) Chambers, D. M.; McElprang, D. O.; Waterhouse, M. G.; Blount, B. C.
Anal. Chem. 2006, 78, 5375–5383
(4) Byfa¨lt Nordqvist, M.; Lo¨f, A.; Osterman-Golkar, S.; Walles, S. A. Chem. Biol.
Interact. 1985, 55, 63–73
(5) Phillips, D. H.; Farmer, P. B. Crit. Rev. Toxicol. 1994, 24, S35–S46
(6) Pauwels, W.; Vodice`ka, P.; Severi, M.; Plna´, K.; Veulemans, H.; Hemminki,
K. Carcinogenesis 1996, 17, 2673–2680
(7) Sepai, O.; Anderson, D.; Street, B.; Bird, I.; Farmer, P. B.; Bailey, E. Arch.
Toxicol. 1993, 67, 28–33
(8) Hemminki, K. Carcinogenesis 1983, 4, 1–3
(9) Yeowell-O’Connell, K.; Pauwels, W.; Severi, M.; Jin, Z.; Walker, M. R.;
Rappaport, S. M.; Veulemans, H. Chem. Biol. Interact. 1997, 106, 67–85
(10) Hemminki, K. Prog. Clin. Biol. Res. 1986, 207, 159–168
(11) Liu, S. F.; Fang, Q. M.; Jin, Z. L.; Rappaport, M. S. J. Environ. Sci. (China)
2001, 13, 391–397
(12) Fustinoni, S.; Colosio, C.; Colombi, A.; Lastrucci, L.; Yeowell-O’Connell, K.;
Rappaport, S. M. Int. Arch. Occup. Environ. Health 1998, 71, 35–41
(13) Liebler, D. C. Chem. Res. Toxicol. 2008, 21, 117–128
.
.
.
.
.
.
Styrene (1) is widely used as an industrial material for the
production of synthetic rubbers, plastic, insulation, fiberglass, and
.
.
* To whom correspondence should be addressed. Jiang Zheng, Center for
Developmental Therapeutics, Seattle Children’s Hospital Research Institute, 1900
Ninth Avenue, Seattle, WA 98101. Phone: (206) 884-7651. Fax: (206) 987-7660.
E-mail: jiang.zheng@seattlechildrens.org.
.
.
.
† Zhejiang University.
‡ University of Washington.
.
§ University of California.
.
2668 Analytical Chemistry, Vol. 81, No. 7, April 1, 2009
10.1021/ac8023262 CCC: $40.75 2009 American Chemical Society
Published on Web 02/26/2009

Scheme 1
adducts modified by reactive metabolites of xenobiotics, such as
atrazine,14 acetaminophen,15 bromobenzene,16 halothane,17 naph-
thalene,18 and trichloroethylene.19 Recently, we developed poly-
clonal antibodies to recognize the proteins modified by racemic
SO.20
specific process by which antibodies are capable of recognizing
antigens stereoselectively.28,29 The purpose of this study was to
develop enantioselective polyclonal antibodies to detect protein
adducts modified by SO enantiomers. These antibody-based
immunoassays are expected to facilitate our investigation on the
biochemical mechanism of styrene-induced toxicity.
Styrene oxide has one chiral center with two optical isomers
as shown in Scheme 1. It was reported that the (R)-styrene oxide
(RSO) was preferentially formed in mice, especially in the lung,21
whereas the (S)-styrene oxide (SSO) was preferentially generated
in rats.22-24 In human volunteers, the cumulative excretion of the
(S)-enantiomer of styrene glycol and mendelic acid were higher
than the R form after exposure to styrene.25 In human liver
microsomes, cytochrome P450-mediated styrene oxidation showed
the production of more SSO relative to RSO.26 It was also found
that SSO was preferentially hydrolyzed than RSO in human liver
microsomes.26 Animal studies showed that the (R)-enantiomer of
SO was more toxic than the (S)-enantiomer in mice.27 The
mechanism of styrene-induced cytotoxicity is not fully understood.
We proposed that the electrophilic epoxide metabolite of styrene
attacks the nucleophilic amino acid residues, such as cysteine, of
cellular proteins (Scheme 1), and the covalent modification of
cellular proteins might initiate the consequent acute toxicity. The
availability of enantioselective antibodies against SO modified
proteins would provide insight into the role of SO chirality in
styrene cytotoxicity both in vitro and in vivo.
EXPERIMENTAL SECTION
Chemicals and Instruments. (R)-styrene oxide (RSO, 98%),
(S)-styrene oxide (SSO, 98%), bovine serum albumin (BSA, 99%),
hemocyanin from keyhole limpet Megathura crenulata (KLH, 5.8
mg/mL PBS solution), horseradish peroxidase (HRP) conjugated
antirabbit IgG secondary antibody, MagicMark XP Western
protein standard, tris(2-carboxyethyl)phosphine (TCEP), and
methyl 3-mercaptopropionate were purchased from Sigma-Aldrich
(St. Louis, MO). N-Succinimidyl(3-(2-pyridyldithio))-propionate
(SPDP), Supersignal West Pico chemiluminescence substrate kit,
and AminoLink Coupling Resin were purchased from Pierce
(Rockford, IL). Other reagents were of analytical grade. BD Falcon
(Bedford, MA) 96-well microtiter plates were used in ELISA. The
absorbance was measured with a microplate reader (VERSAMax
,
Molecular Devices, Sunnyvale, CA). The ELISA absorbance
data were analyzed by SigmaPlot for Windows 9.0. SDS-PAGE
was performed on a Bio-Rad mini electrophoresis system.
Membrane transfer was conducted on a Bio-Rad Trans-Blot SD
Semi-Dry Electrophoretic Transfer Cell. Structural identification
of synthetic compounds was performed on a 300 MHz NMR
spectrometer (Varian Associates, Palo Alto, CA) and an LC/
MS system including an Agilent 1100 HPLC system interfaced
with a Sciex API 2000 tandem quadrupole mass spectrometer
(Applied Biosystems, Foster City, CA).
Synthesis. Immunogens (Thiolated KLH-RSO/SSO Adducts,
5 and 6). Immunogens were synthesized as illustrated in Scheme
2, using the method reported by our laboratory18,20 with minor
modification. SPDP (4.5 mg dissolved in 150 µL of dimethyl
sulfoxide (Me2SO)) was added to 6.0 mL of KLH solution (5.8
mg/mL) dropwise while stirring. After stirring for 1.5 h at room
temperature, TCEP (52.6 mg dissolved in 300 µL of PBS) was
added to this mixture dropwise with stirring. After stirring for
3 h at room temperature under nitrogen, the pH of the mixture
was adjusted to 9-10 with diluted NaOH solution, and then
the mixture was divided into two aliquots. To each aliquot, 30
µL of RSO or SSO diluted in 150 µL of Me2SO was added
dropwise with stirring. The mixture was stirred overnight at
Immunoassay offers a powerful tool to investigate the toxico-
logical importance of protein modification in experimental animals
and exposed humans. Antibody-antigen recognition is a highly
(14) Dooley, G. P.; Hanneman, W. H.; Carbone, D. L.; Legare, M. E.; Andersen,
M. E.; Tessari, J. D. Chem. Res. Toxicol. 2007, 20, 1061–1066
(15) Pumford, N. R.; Roberts, D. W.; Benson, R. W.; Hinson, J. A. Biochem.
Pharmacol. 1990, 40, 573–579
(16) Rombach, E. M.; Hanzlik, R. P. Chem. Res. Toxicol. 1999, 12, 159–163
(17) Martin, J. L.; Meinwald, J.; Radford, P.; Liu, Z.; Graf, M. L.; Pohl, L. R.
Drug Metab. Rev. 1995, 27, 179–189
(18) Zheng, J.; Hammock, B. D. Chem. Res. Toxicol. 1996, 9, 904–909
(19) Halmes, N. C.; McMillan, D. C.; Oatis, J. E., Jr.; Pumford, N. R. Chem. Res.
Toxicol. 1996, 9, 451–456
(20) Yuan, W.; Chung, J.; Gee, S.; Hammock, B. D.; Zheng, J. Chem. Res. Toxicol.
2007, 20, 316–321
(21) Carlson, G. P. J. Toxicol. Environ. Health 1997, 51, 177–187
(22) Foureman, G. L.; Harris, C.; Guengerich, F. P.; Bend, J. R. J. Pharmacol.
Exp. Ther. 1989, 248, 492–497
(23) Watabe, T.; Ozawa, N.; Yoshikawa, K. Biochem. Pharmacol. 1981, 30, 1695–
1698
(24) Battistini, C.; Bellucci, G.; Mastrorilli, E. Xenobiotica 1979, 9, 57–61
(25) Wenker, M. A.; Kezic´, S.; Monster, A. C.; de Wolff, F. A. Int. Arch. Occup.
Environ. Health 2001, 74, 359–365
(26) Wenker, M. A.; Kezic´, S.; Monster, A. C.; de Wolff, F. A. Toxicol. Appl.
Pharmacol. 2000, 169, 52–58
(27) Gadberry, M. G.; DeNicola, D. B.; Carlson, G. P. J. Toxicol. Environ. Health
1996, 48, 273–294
.
.
.
.
.
.
.
.
.
.
.
.
.
(28) Hofstetter, H.; Hofstetter, O. Trends Anal. Chem. 2005, 24, 869–879
(29) Got, P. A.; Scherrmann, J. M. Pharm. Res. 1997, 14, 1516–1523
.
.
.
Analytical Chemistry, Vol. 81, No. 7, April 1, 2009 2669

Scheme 2
Scheme 3
against deionized water and dried by lyophilization separately.
In order to prepare BSA-RSO and BSA-SSO with a variety
of hapten loading, BSA (10 mg/mL) was incubated with RSO
or SSO at various concentrations (1.0, 5.0, and 10 mM) in PBS
buffer (pH 7.4, 50% of Me2SO) with stirring overnight at 4 °C.
A similar procedure of dialysis and lyophilization was performed
as above.
Methyl 3-(2-hydroxyphenylethylthio)propanoates (MHPPs, 7-10).
Compounds 7-10 were synthesized as competitors (analytes)
for competitive ELISA and Western blot. They mimic the epitopes
of protein adducts by SO at cysteine residues. Enantiomeric SO
(360 mg, 3 mmol), either RSO or SSO, was dissolved in 5 mL of
THF/water (10:1), followed by addition of methyl 3-mercaptopro-
pionate (372 mg, 3.1 mmol) and triethylamine (909 mg, 9 mmol).
The reaction mixture was stirred at room temperature overnight,
and then most of the solvent was removed under reduced
pressure. The residue was extracted with ethyl acetate (10 mL ×
3), and the combined organic phase was washed with saturated
sodium bicarbonate solution and brine, dried over anhydrous
sodium sulfate, and concentrated under a vacuum. The residue
was chromatographed on silica gel to give the corresponding
methyl 3-(2-hydroxy-2-phenylethylthio)propanoate (MH2PP (7/
9), 281 mg, 39%) and methyl 3-(2-hydroxy-1-phenylethylthio)pro-
panoate (MH1PP (8/10), 187 mg, 26%). The structures of the
synthetic competitors were confirmed by NMR. MH2PP, 1H NMR
(CDCl3): δ 2.63 (m, 2 H), 2.77-2.98 (m, 4H), 3.73 (s, 3H),
4.78-4.82 (m, 1H), 7.32-7.40 (m, 5H). MH1PP, 1H NMR
(CDCl3): δ 2.52-2.57 (t, J ) 7.5 Hz, 2 H), 2.71-2.77 (m, 2H),
3.69 (s, 3H), 3.88-3.91 (m, 2H), 3.99-4.04 (m, 1H), 7.30-7.37
(m, 5H).
room temperature under nitrogen. The next day, the products
were dialyzed five times against 1 L of deionized water and
lyophilized separately. Treatment of the thiolated KLH with
RSO or SSO gave the (R)-immunogen and (S)-immunogen,
respectively.
Verification of Synthetic Immunogens. In order to ensure
success in the synthesis of the immunogens, i.e. (R)-immunogen
and (S)-immunogen, we characterized the protein adducts as
follows. The lyophilized immunogens (5.0 mg) were separately
suspended in 4.0 mL of propylamine (12), followed by addition
of 18 M NaOH solution (0.5 mL). The mixture was heated in a
tightly closed tube with stirring at 80 °C in an oil bath for 4 h.
After the mixture was cooled down to room temperature, the
excess of propylamine was evaporated under a stream of nitrogen.
The remaining was resuspended in 200 µL of acetonitrile, vortexed
for 3 min, and centrifuged at 12 000 rpm for 10 min. The
supernatant was collected and subjected to LC/MS analysis. MS
analysis was performed in selective ion monitoring (SIM) mode
to monitor m/z 268 [M + H]+ (Scheme 3). The control sample
was treated by the same procedure without exposure to SO.
BSA-RSO and BSA-SSO Adducts. Protein adducts, bovine
serum albumin-(R)-styrene oxide (BSA-RSO) and bovine serum
albumin-(S)-styrene oxide (BSA-SSO), were synthesized as
coating antigens ((R)- and (S)-coating antigens) for ELISA and
positive control in Western blot analysis. To 2 mL of BSA solution
(10 mg/mL in PBS buffer, pH 7.4), 20 µL of RSO or SSO dissolved
in 100 µL of Me2SO was added and stirred overnight at room
temperature. The resulting mixtures were extensively dialyzed
Methyl 3-(2-phenylethylthio)propanoate (MPP, 11). A mixture
of 2-phenylethanethiol (138 mg, 1.0 mmol) and methyl acrylate
(95 mg, 1.1 mmol) was stirred at room temperature until TLC
analysis showed that most of 2-phenylethanethiol was consumed,
and then the reaction mixture was subject to silica gel chroma-
tography without further workup to give MPP (11, 96 mg, 42.8%)
as a colorless oil. MPP, 1H NMR (CDCl3): δ 2.61-2.66 (t, J )
2670 Analytical Chemistry, Vol. 81, No. 7, April 1, 2009

7.5 Hz, 2H), 2.81-2.84 (m, 4H), 2.89-2.94 (m, 2H), 7.21-7.35
(m, 5H).
Competitive ELISA. Totally five competitors (7-11) were
used in the competitive ELISA (Table 1). A volume of 100 µL of
coating antigen solution (20 µg/mL of BSA-RSO or BSA-SSO
in PBS) was added to each well of 96-well microtiter plates and
incubated at 4 °C overnight. Serial dilutions of the competitors
(MHPPs or MPP, 1-10-7 mM except for that of (R)-MH2PP,
i.e., 10-10-6 mM) were prepared in diluted antisera (1:3200)
in 5% nonfat milk-PBST buffer. The resulting mixtures were
incubated at 4 °C overnight. The following day, the same
procedure in the titer analysis was followed. The absorbance
at dual wavelengths (450-650 nm) was read. The obtained
absorbance values were converted using the equation: control
(%) ) A/A0 × 100%, where A represents the absorbance values
in the presence of a competitor and A0 is the absorbance
without a competitor. The resulting competition curves were
fitted by a four-parameter logistic equation using SigmaPlot.
IC50 values (the concentration that produced 50% inhibition)
were obtained from the regression curve. The cross activity
was defined as the ratio of IC50 from the same antiserum to
the corresponding enantiomer.
Western Blot Analysis. A total of 2 µg of protein was loaded
and resolved by SDS-PAGE. The protein bands resolved were then
transferred to a PVDF membrane using a semidry transfer cell
(Bio-Rad) at 15 V for 30 min. Following transfer, the membrane
was washed in Tris-buffered saline with Tween-20 (TBST, 10 mM
Tris, 0.15 M NaCl, 0.05% Tween-20, pH 7.5) once and blocked
with 5% nonfat milk-TBST buffer for 1 h at room temperature.
After the membrane was blocked, it was incubated with antiserum
no. 1254 (antiserum against the (R)-immunogen, 1:2 000) in 5%
nonfat milk-TBST buffer overnight at 4 °C. The next day, the
membrane was washed with TBST four times for 2 min and then
incubated with a HRP conjugated antirabbit antibody at a 1:10 000
dilution in 5% nonfat milk-TBST buffer for 1 h at room temper-
ature. The membrane was again washed with TBST buffer four
times, and the protein bands were detected by Supersignal West
Pico chemiluminescence kit and visualized using a Perkin-Elmer
Geliance 600 imaging system (Perkin-Elmer, Fremont, CA).
Immunoblot treated with antiserum no. 1257 (antiserum against
the (S)-immunogen, 1:2 000) was performed in parallel.
Exposure of WI-38 Cells to Styrene Oxide Enantiomers.
Human lung cell line WI-38 cells were cultured in RPMI 1640
medium (Mediatech Inc., Manassas, VA) supplemented 10% FBS
(HyClone, Logan, UT). WI-38 cells were seeded on 6-well plates
at a density of 2 × 105 cells per well. The culture medium was
discarded after being cultured for 24 h, and fresh medium
containing 0.5 mM or 2.0 mM RSO or SSO was supplemented.
The cells were cultured for another 24 h, and the cells were
harvested by digestion and washed with PBS three times. The
cells were lysed by sonication on ice for 1 min. The protein
concentration of the cell lysate was determined by the BCA
protein determination kit.
Immobilization of BSA-RSO and BSA-SSO to Agarose
Beads. AminoLink Coupling Resin (Pierce, Rockford, IL) was
utilized as a beaded agarose support. BSA (20 mg in 2 mL buffer)
was immobilized to 2 mL of the resin following the manufacturer’s
instruction. By comparison of the BSA concentration before and
after immobilization, the coupling efficiency was found to be about
80%. After immobilization of BSA, 10 µL of RSO or SSO dissolved
3-(2-Hydroxy-2-phenylethylthio)-N-propylpropanamide (HPPPA,
13). MH2PP (7/9, 50 mg, 0.2 mmol) was dissolved in 1.0 mL
methanol, followed by addition of 3.0 mL of propylamine and
stirred at room temperature until TLC analysis showed that most
of MH2PP was consumed. Then the reaction mixture was subject
to silica gel chromatography without further workup to give
HPPPA (35 mg, 67%). HPPPA, 1H NMR (CDCl3): δ 0.88-0.93
(t, J ) 7.4 Hz, 3H), 1.46-1.56 (m, 2H), 2.40-2.53 (m, 2H),
2.72-2.93 (m, 4H), 3.15-3.21 (m, 2H), 4.76-4.80 (m, 1H), 6.14
(s, 1H), 7.28-7.38 (m, 5H). LC/MS: m/z 268 [M + H]+.
Immunization of Rabbits. Six female New Zealand white
rabbits (Herbert’s Rabbitery, Plymouth, CA) weighing 2.5-3.0 kg
were divided into two groups and immunized with (R)- and (S)-
immunogens, respectively. Each immunogen (100 µg) was dis-
solved in 0.5 mL of PBS buffer (pH 7.4) and emulsified with 0.5
mL of Freund’s complete adjuvant. The rabbits were injected
subcutaneously with the emulsion (1.0 mL/rabbit) at multiple sites
on the back. The rabbits were boosted with a 2 week interval by
the same procedure except that the Freund’s complete adjuvant
was replaced by Freund’s incomplete adjuvant. Test bleeds were
collected before each boosting injection, and the titer and
enantioselectivity was examined by ELISA. The rabbits were
boosted until no further increase in antibody titer was observed.
Analysis of Antiserum Titer. The titer of the antisera
obtained from the rabbits immunized with (R)-immunogen or (S)-
immunogen was determined by measuring the binding of serial
dilutions (1:400 to 1:12 800) to microtiter plates coated with coating
antigens BSA-RSO, BSA-SSO, or native BSA. Coating antigen
solution (100 µL, 20 µg/mL) in PBS (10 mM phosphate-buffered
saline, pH 7.4, 0.15 M NaCl) was added to each well of 96-well
microtiter plates and incubated at 4 °C overnight. The coating
antigen solution was discarded, and the plates were washed three
times with PBST (10 mM phosphate-buffered saline, pH 7.4, 0.15
M NaCl, 0.05% Tween-20). After washing, 150 µL of 5% nonfat
milk-PBST buffer was added to each well and incubated at room
temperature for 1.5 h. The plates were washed three times with
PBST buffer after incubation, and 100 µL of serially diluted
antiserum in PBST was added to each well. After incubation at
room temperature for 2 h, the plates were washed three times in
the same manner, and 100 µL of HRP conjugated secondary
antibody (antirabbit IgG, 1:10 000) in PBST was added to each
well. After incubation at room temperature for 1 h, the plates were
washed three times as before, and 100 µL of substrate solution
(0.3 mM tetramethylbenzidine, 0.4 mM H2O2 in 0.1 M sodium
acetate buffer at pH 5.5) was added to each well. After
incubation for 15 min at room temperature, the colorimetric
development was quenched by adding 50 µL of 4 N H2SO4
solution to each well. The absorbance at dual wavelengths
(450-650 nm) was measured. To evaluate its affinity, each
antiserum was tested on all three coating antigens. The titration
curves were fitted by four-parameter logistic equation using
SigmaPlot. The enantioselectivity index was defined as the ratio
of EC50 (antiserum dilution which produced 50% highest
absorbance) obtained from (R)- or (S)-coating antigens (REC50
or SEC50).
Analytical Chemistry, Vol. 81, No. 7, April 1, 2009 2671

Table 1. Structures of Analytes, IC50 Values, and Calculated Cross Activity of Antisera Numbers 1254 and 1257
in 100 µL of Me2SO was added to the resin with 1 mL of 0.1 M
PBS at pH 9.2 and mixed overnight at 4 °C. The resulting resin
was packed into a column and washed with 10 mL of 0.1 M
PBS pH 7.2 for use.
(SO) as a major metabolite is associated with the cytotoxicity of
styrene. We proposed that protein modification by SO may play
an important role in styrene toxicity. Recently, we prepared a
polyclonal antibody as an experimental tool to detect racemic SO
modified proteins.20 The effort in the development of antibodies
to selectively detect protein adducts modified by SO enantiomers
was inspired by the findings of toxic effects of SO with enantio-
meric differentiation.19-25 The availability of the antibodies would
assist us to investigate the enantiomeric role of SO in styrene-
mediated toxicity. A number of immunoassays developed for
analysis of chiral drugs, such as propanolol,30 alprenolol,31 and
pentobarbital,32 ractopamine,33 finrozole,34 and S-20499,35 have
been reported. Some efforts have been made to develop enanti-
Purification of Antibodies. In order to enhance the enan-
tioselectivity of the polyclonal antibodies, the antisera were
purified by use of the affinity columns prepared above. Briefly,
antiserum no. 1254 or no. 1257 (10 µL) was diluted in 0.5 mL
of 0.1 M PBS (pH 7.2) and slowly loaded onto the affinity
column. After standing at room temperature for 2 h, the column
was eluted with 12 mL of 0.1 M PBS and fractions were
collected. The enantioselectivity of each fraction was evaluated
by titration analysis. The column was regenerated by elution
with 8 mL of 0.1 M glycine-HCl (pH 2.5) buffer and then 16
mL of 0.1 M PBS (pH 7.2).
(30) Wang, L.; Chorev, M.; Feingers, J.; Levitzki, A.; Inbar, M. FEBS Lett. 1986,
199, 173–178
(31) Chamat, S.; Hoebeke, J.; Strosberg, A. D. J. Immunol. 1984, 133, 1547–
1552
(32) Cook, C. E.; Seltzman, T. B.; Tallent, C. R.; Lorenzo, B.; Drayer, D. E.
J. Pharmacol. Exp. Ther. 1987, 241, 779–785
(33) Weilin, L. S.; David, J. S.; Eugene, S. B. J. Agric. Food Chem. 2000, 48,
4020–4026
.
.
RESULTS AND DISCUSSION
.
The biochemical mechanism of styrene-induced cytotoxicity
has not been fully understood. It was suggested that styrene oxide
.
2672 Analytical Chemistry, Vol. 81, No. 7, April 1, 2009

Figure 1. LC/MS profiles of (R)-immunogen (B) and thiolated KLH (C) after reaction with propylamine at 80 °C for 4 h. LC/MS profile of
authentic synthetic 3-(2-hydroxy-2-phenylethylthio)-N-propylpropanamide (A).
oselective antibodies to detect protein adducts modified by chiral
ligands, such as ibuprofen.36 The present study is a follow-up of
our earlier antibody work designed to detect protein SO (racemic)
adducts.
(13, Scheme 3), which can be readily detected by LC/MS. HPPPA
was synthesized by conjugating methyl 3-mercaptopropionate with
SO, followed by reaction with propylamine. Immunogens, includ-
ing the KLH adducts modified by either SSO or RSO, were treated
with propylamine and analyzed by LC/MS in the SIM mode.
Thiolated KLH as a negative control was treated in parallel. Figure
1 shows the ion chromatography profiles for m/z 268, and a peak
at 8.4 min was observed in the sample obtained from the
immunogens after treatment with propylamine. The retention time
of the peak (Figure 1B) was found to be identical to that of
authentic standard 13 (Figure 1A). As expected, no such peak
was detected in the control sample (Figure 1C). The observed
amide 13 indicates the incorporation of the hapten into the carrier
protein. Although this approach was unable to provide quantitative
information about hapten loading due to the lacking of reaction
yields, it allowed us to verify the hapten incorporation into the
protein in identity. The assurance of the chemical identities of
immunogens was an important prerequisite for the success in the
preparation of antibodies.
Analysis of Titer. Immunizations of rabbits were carried out
with (R)- and (S)-immunogens. The titers of the antisera
obtained from the rabbits were determined by measuring the
binding of serial dilutions (1:400 to 1:12 800) to microtiter plates
coated with BSA-RSO, BSA-SSO, or native BSA. The enanti-
oselectivity of the antibodies was evaluated by the degree of
cross-reaction to the counter-enantiomeric coating antigens.
The results showed that all six rabbits gave very high titers of
antibodies against their own enantiomeric coating antigens
accordingly. The three rabbits (numbers 1252, 1253, and 1254)
immunized with the (R)-immunogen produced enantioselective
Synthesis and Verification of the Immunogens. Immuno-
gens were synthesized following an earlier procedure by which
we succeeded in raising polyclonal antibodies against protein
adducts modified by SO racemates. Carrier protein KLH was first
thiolated by SPDP, and the resulting thiolated protein was reduced
by TCEP to generate free sulfhydryl groups, which immediately
reacted with RSO or SSO, respectively (Scheme 2). We learned
from our previous work that the thiolation of KLH allowed us to
incorporate a spacer between the carrier protein and the hapten
to increase the visibility of the hapten.
Verification of the structure of immunogens is an important
step to succeed in development of antibodies and is often a
challenge in immunochemistry. In the present study, we success-
fully developed a chemical approach to characterize the incorpora-
tion of the hapten to the carrier protein. As shown in Scheme 2,
the thiolation of the carrier protein by reacting with SPDP
introduces an amide. This provides the possibility to release the
carboxyl moiety by transamidation. The transamidation reaction
was performed by heating the immunogens in neat propylamine
(12), and the reaction was expected to produce amide HPPPA
(34) Nevanen, T. K.; So¨derholm, L.; Kukkonen, K.; Suortti, T.; Teerinen, T.;
Linder, M.; So¨derlund, H.; Teeri, T. T. J. Chromatogr., A 2001, 925, 89–
97.
(35) Got, P. A.; Guillaumet, G.; Boursier-Neyret, C.; Scherrmann, J. M. J. Pharm.
Sci. 1997, 86, 654–659.
(36) Ito, H.; Ishiwata, S.; Kosaka, T.; Nakashima, R.; Takeshita, H.; Negoro, S.;
Maeda, M.; Ikegawa, S. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci.
2004, 806, 11–17
.
Analytical Chemistry, Vol. 81, No. 7, April 1, 2009 2673

Figure 2. Titer analysis of antisera numbers 1254 (A) and 1257 (B) in microtiter plates coated with BSA-RSO (b), BSA-SSO (O), or native
BSA (2). Curves were fitted by four-parameter logistic equation using SigmaPlot. Error bars indicate standard deviations of triplicate determinations.
Figure 3. Titer analysis of antiserum no. 1254 before (A) and after (B) purification and antiserum no. 1257 before (C) and after (D) purification.
Titration was performed in microtiter plates coated with BSA-RSO (b) or BSA-SSO (O).
antibodies (Figure S-1 in the Supporting Information), while
only one rabbit (no. 1257) among the three of the (S)-group
gave enantioselective antibodies (Figure S-1 in the Supporting
Information). The antisera obtained from the six rabbits showed
little cross-reaction to native BSA. Among the rabbits im-
munized with the (R)-immunogen, rabbit no. 1254 produced
the antiserum with highest titer and enantioselectivity. Figure
2A shows the titration of antiserum no. 1254 against (R)- and
(S)-coating antigens as well as native BSA. The enantioselec-
tivity index (REC50/SEC50) of antiserum no. 1254 was
estimated to be 10. Similarly, Figure 2B shows the titration
of antiserum obtained from rabbit no. 1257 immunized with
the (S)-immunogen. The enantioselectivity index (SEC50/
REC50) for antiserum no. 1257 was approximately 5. Because
of the higher titer and enantioselectivity, antisera numbers
1254 and 1257 were selected for use in the remainder of the
studies.
Competitive ELISA. Competitive ELISA was conducted to
characterize the hapten-selectivity and enantioselectivity of the
antibodies. Antisera numbers 1254 and 1257 raised against
respective (R)- and (S)-immunogen were diluted to 1:3 200 and
used for competitive ELISA in plates coated with the correspond-
ing coating antigens as those for the titration tests. A total of four
SO derivatives, including MHPPs 7-10, and MPP were employed
2674 Analytical Chemistry, Vol. 81, No. 7, April 1, 2009

Figure 4. Western blot analysis of native BSA, BSA-RSO, BSA-SSO, BSA-RSO/BSA 1:1 mixture, BSA-SSO/BSA 1:1 mixture, BSA-RSO
and BSA-SSO 1:1 mixture. Protein bands were incubated with antisera no. 1254 (R, 1:2000) and no. 1257 (S, 1:2000). Each lane was loaded
with 2.0 µg of protein.
Figure 5. Western blot analysis of BSA exposed to various concentrations (1.0, 5.0, and 10 mM) of RSO or SSO. Protein adducts bands were
treated with antisera no. 1254 (R, 1:2000) and no. 1257 (S, 1:2000). Each lane was loaded with 2.0 µg of protein.
as competitors (analytes). The antisera were preincubated with a
serial dilution of the synthetic competitors.
interactions between the antibodies and the haptens. Importantly,
this provides strong evidence for the success in the preparation
of the antibodies we desired. The immuno-recognition pinpointed
the core of the haptens.
Table 1 lists the IC50 values obtained from the competitive
ELISA analysis. As expected, the preincubation of antisera with
MHPPs 7/8 and 9/10 showed inhibitory effects on the
binding of the antibodies to the corresponding coating antigens
(Figure S-2 in the Supporting Information). Compounds 7/9
and 8/10 are two pairs of enantiomers derived from RSO and
SSO, respectively. The affinity of the two pairs to the antibodies
allowed us to evaluate the enantioselectivity of the antisera raised
from (R)- and (S)-immunogens. Antiserum no. 1254 was prein-
cubated with a serial dilution of the analytes in microtiter plates
coated with (R)-coating antigen, and antiserum no. 1257 was
treated similarly but with (S)-coating antigen. As expected,
antiserum no. 1254 showed higher affinity to analytes 7 and 8
than 9 and 10 accordingly. However, antiserum no. 1257 showed
higher affinity to analytes 9 and 10 than 7 and 8. This was the
additional evidence for the enantiomeric recognition of chiral SO-
derived haptens by antisera numbers 1254 and 1257.
Compounds 7/8 and 9/10 are two pairs of positional isomers.
The positional isomers of each pair revealed differentiated inhibi-
tion of the antibody-coating antigen binding (Table 1). Interest-
ingly, isomers 8 and 10 were found to be more potent than
isomers 7 and 9, respectively. Isomers 8 and 10 were derived
from RSO and SSO with conjugation at the R-carbon, and isomers
7 and 9 were derived from the conjugation at the ꢀ-carbon. Our
earlier studies found that the reaction of SO with N-acetyl cysteine
produced a 2:1 mixture of the R-carbon conjugate over the
ꢀ-carbon conjugate.20 It is likely that similar preferred nucleophilic
conjugation took place when SO reacted with thiolated KLH. This
may explain why the polyclonal antibodies revealed higher affinity
toward competitors 8/10 rather than 7/9.
Purification of Antiserum. Affinity purification is a conven-
tional and useful method to purify antibodies, and we examined
whether affinity chromatography would significantly improve the
enantioselectivity of the antibodies. Protein adducts BSA-RSO
and BSA-SSO were coupled to the AminoLink Coupling Resin
Among the five analytes tested, only MPP failed to inhibit the
binding of the antibodies to the coating antigens. This indicates
the critical role of the hydroxyl group of the haptens in the
Analytical Chemistry, Vol. 81, No. 7, April 1, 2009 2675

Figure 6. Western blot analysis of BSA adducts exposed to a selection of concentrations (1.0, 5.0, and 10 mM, left) of RSO or SSO, and BSA
adduct (after exposure to 10 mM of RSO or SSO) diluted with native BSA (right). BSA-RSO bands were incubated with antiserum no. 1254 (R,
1:2000) and BSA-SSO bands were incubated with antisera no. 1257 (S, 1:2000). Each lane was loaded with 2.0 µg of protein.
Figure 7. Competitive Western blot analysis of BSA-RSO and BSA-SSO adducts. BSA-RSO bands were incubated with antiserum no.
1254 (R, 1:2000) mixed with (R)-MH1PP at concentrations of 0, 0.01, 0.1, and 1.0 mM. BSA-SSO bands were incubated with antiserum no.
1257 (S, 1:1000) together with (S)-MH1PP at concentrations of 0, 0.01, 0.1, and 1.0 mM. Each lane was loaded with 2.0 µg of protein.
individually as described in the experimental procedure. The
undesired antibodies were retained on the affinity columns, and
the desired antibodies were eluted out. The titers of the purified
antisera were evaluated by titration tests. Figure 3 shows the
titrations of antisera numbers 1254 and 1257 before and after the
affinity purification. The results showed that their enantioselec-
tivity was appreciably improved, but the purification system had
limited capacity. It appears that it is not realistic to obtain
enantioselective antibody from racemic antibodies by means of
affinity chromatography.
Western Blot Analysis. The enantioselectivity of antisera
numbers 1254 and 1257 was assessed by Western blot analysis.
As shown in Figure 4, a chemiluminescent band at the molecular
weight of BSA was observed in the lane loaded with BSA-RSO
adduct incubated with antiserum no. 1254. As expected, no
chemiluminescent band was observed in the lane loaded with
native BSA. This suggests that the antibodies are able to detect
BSA adduct modified with SO and show no cross-reaction toward
native BSA. In the same experiment, BSA-SSO adduct was loaded
in SDS-PAGE for immunoblot analysis, and no bands with
significant chemiluminescence were observed. This indicates little
cross-reaction of the antiserum raised from the (R)-immunogen
toward the (S)-antigen, BSA-SSO adduct. Similarly, antiserum
no. 1257 showed no cross-reaction toward native BSA and minor
affinity to the (R)-antigen, BSA-RSO adduct. These immunoblot
results clearly indicate that antisera numbers 1254 and 1257 can
differentiate the chirality of the haptens and provide high enan-
tioselectivity of immunorecognition.
In an effort to evaluate the correlation between antibody
binding and hapten loading in BSA-RSO and BSA-SSO adducts,
these adducts with various hapten loading were synthesized by
exposure of BSA to RSO or SSO at concentrations of 1, 5, and 10
mM, respectively. After extensive dialysis, the resulting BSA-RSO
and BSA-SSO adducts were subjected to Western blot analysis.
As expected, the higher the hapten load, the greater the
luminescent intensity in the immunoblot (Figure 5), indicating
that the strength of antibody recognition depended on the hapten
loading of the protein adducts.
2676 Analytical Chemistry, Vol. 81, No. 7, April 1, 2009

At the same time, the BSA-RSO and BSA-SSO adducts
synthesized by exposure to 10 mM of SO enantiomer were diluted
with native BSA at ratios of 1:9 and 1:1 and were analyzed by
Western blot. The dilution ratios corresponded to the concentra-
tions of SO enantiomers (1, 5, 10 mM) used the preparation of
BSA-RSO and BSA-SSO adducts. As shown in Figure 6, the
pattern of luminescent bands was found similar to that observed
in the Western blot of BSA exposed to various concentrations of
SO enantiomers. The BSA-RSO and BSA-SSO adducts without
native BSA dilution produced the highest luminescent intensity,
followed by those diluted with 2- and 10-fold BSA. This suggested
a good quantitative immunoresponse of the antibodies to the
protein adducts.
Competitive immunoblot analysis was conducted to determine
the hapten selectivity of the antibodies. The membrane blotted
with BSA-RSO and BSA-SSO adducts was cut into four pieces
and incubated with antisera numbers 1254 and 1257 containing a
series of concentrations (0, 0.01, 0.1, and 1 mM) of (R)-MH1PP,
8, for antiserum no. 1254 or (S)-MH1PP, 10, for antiserum no.
1257, respectively. The resulting membranes were incubated with
the secondary antibody and treated with the chemiluminescence
kit as above. As shown in Figure 7, the chemiluminescent intensity
decreased with the increasing of the concentrations of the
competitors coincubated with the corresponding antisera. This
indicates that the binding of the antibodies to SO protein adducts
is attributable to the antibody recognition of the SO-protein
modification at cysteine residues. We did not test the effects of
(R)-MH2PP and (S)-MH2PP in the competitive immunoblot
analysis, since competitive ELISA experiments above showed that
the affinity of (R)-MH2PP and (S)-MH2PP to the corresponding
antibodies was much weaker than that of (R)-MH1PP and (S)-
MH1PP.
Western Blot Analysis of Cellular Protein Modified by
Styrene Oxide Enantiomer. To examine the ability of the
antisera to detect SO modified cellular protein, WI-38 cellular
protein modified by RSO or SSO were subjected to Western blot
analysis. The blotted protein was detected by corresponding
numbers 1254 or 1257 antiserum (1:1000), respectively. Several
new bands were detected after exposure to RSO, and high-density
bands were detected in cellular protein exposed to high concen-
tration (Figure S-3 in the Supporting Information). The result
showed the antibodies can recognize cellular protein modified by
SO, and it was concentration dependent.
In summary, we have successfully developed polyclonal
antibodies to recognize chiral SO-derived protein (BSA) adducts
modified at cysteine residues with no cross reaction toward native
BSA. The antibodies revealed high enantioselectivity of immu-
norecognition, providing a powerful tool for the investigation of
the biochemical mechanism of cytotoxicity induced by styrene.
ACKNOWLEDGMENT
We would like to thank Dr. Sihoun Hahn’s laboratory for their
technical support on the Western blot analysis. This work was
supported by NIH Grant HL080226 and NIEHS Superfund Basic
Research Program P42 ES04699.
SUPPORTING INFORMATION AVAILABLE
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
Received for review November 4, 2008. Accepted January
30, 2009.
AC8023262
Analytical Chemistry, Vol. 81, No. 7, April 1, 2009 2677