Immunochemical detection of food-derived isothiocyanate as a lysine conjugate

Article abstract of DOI:10.1271/bbb.90728

In a previous study we prepared monoclonal antibody against allyl isothiocyanate (AITC)-modified lysine (Lys), and found that AITC reacted with Lys under physiological conditions in vitro (T. Nakamura et al., Chem. Res. Toxicol., 22, 536-542 (2009)). In the present study, antibodies against benzyl isothiocyanate (ITC), 6-methylsulfinylhexyl ITC and phenethyl ITC modified protein were prepared, and the respective monoclonal antibodies, B6C9, 6MS3D10, and PE3A10 were obtained. These antibodies were applied to ITC detection in food using shredded Wasabia japonica (wasabi) and ground Carica papaya (papaya) seed by trapping ITC with biotin-labeled bovine serum albumin. ITC formation from the wasabi and papaya seed samples was confirmed using the antibodies in a dose-dependent manner. These antibodies might be applicable in identifying food-derived ITC.

Full text of DOI:10.1271/bbb.90728

Biosci. Biotechnol. Biochem., 74 (3), 536–540, 2010
Immunochemical Detection of Food-Derived Isothiocyanate
as a Lysine Conjugate
y
1;
Toshiyuki NAKAMURA,1 Noritoshi KITAMOTO,1 Toshihiko OSAWA,2 and Yoji KATO
1Graduate School of Human Science and Environment, University of Hyogo, Himeji 670-0092, Japan
2Nagoya University Graduate School of Bioagricultural Sciences, Nagoya 464-8601, Japan
Received October 5, 2009; Accepted December 3, 2009; Online Publication, March 7, 2010
[doi:10.1271/bbb.90728]
In a previous study we prepared monoclonal antibody
against allyl isothiocyanate (AITC)-modified lysine
(Lys), and found that AITC reacted with Lys under
physiological conditions in vitro (T. Nakamura et al.,
Chem. Res. Toxicol., 22, 536–542 (2009)). In the present
study, antibodies against benzyl isothiocyanate (ITC),
6-methylsulfinylhexyl ITC and phenethyl ITC modified
protein were prepared, and the respective monoclonal
antibodies, B6C9, 6MS3D10, and PE3A10 were ob-
tained. These antibodies were applied to ITC detection
in food using shredded Wasabia japonica (wasabi) and
ground Carica papaya (papaya) seed by trapping ITC
with biotin-labeled bovine serum albumin. ITC forma-
tion from the wasabi and papaya seed samples was
confirmed using the antibodies in a dose-dependent
manner. These antibodies might be applicable in
identifying food-derived ITC.
tandem mass spectrometry.5) 6MSITC and its thiol
conjugate were detected by HPLC-atmospheric pressure
chemical ionization mass spectrometry.6) Methods of
ITC detection in food and in biological samples have
been limited to these chemical analyses to date.
Recently, we prepared an antibody to AITC-modified
lysine (Lys).7) In addition to this antibody, in the current
study we prepared monoclonal antibodies against food-
derived ITC-modified protein using BITC, 6MSITC, and
PEITC. Using these antibodies with biotin-conjugated
bovine serum albumin (BSA), the formation of ITC
from ground food was confirmed.
Materials and Methods
Materials. AITC, BITC, and PEITC were purchased from Tokyo
Chemical Industry (Tokyo, Japan). 6MSITC and 6MTITC were kindly
donated by Kinjirushi (Nagoya, Japan). S-(N-Benzylthiocarbamoyl)-L-
cysteine (BITC-Cys) was purchased from LKT Laboratories (St. Paul,
MN). BSA (A7511, ꢀ97%), biotinamidocaproyl labeled BSA (biotin-
BSA), and streptavidin-peroxidase polymer (ultrasensitive) were from
Sigma-Aldrich (St. Louis, MO). Keyhole limpet hemocyanin (KLH)
was from Pierce (Rockford, IL). Nꢀ-Benzoyl-glycyl-L-lysine (BGK)
was from Peptide Institute (Osaka, Japan). Nꢀ-Acetyl-L-cysteine
(NAC) was from Nacalai Tesque (Kyoto, Japan). All other chemicals
were purchased from Wako Chemical (Osaka, Japan).
Key words: isothiocyanate; lysine adduct; antibody;
enzyme-linked immunosorbent assay
Isothiocyanates (ITCs) are present in cruciferous
plants as glucosinolates, and are produced by myrosinase
when plant cells are injured. Wasabia japonica (wasabi),
Carica papaya (papaya), and water cress are sources of
allyl ITC (AITC), benzyl ITC (BITC), and phenethyl
ITC (PEITC) respectively (Fig. 1). Other known
characteristic wasabi compounds include 6-methylsulfi-
nylhexyl ITC (6MSITC) and 6-methylthiohexyl ITC
(6MTITC) (Fig. 1).
ITC reacts preferentially with thiols, forming a
dithiocarbamate derivative. ITCs and their dithiocarba-
mates react quantitatively with 1,2-benzenedithiol by a
cyclocondensation reaction to give rise to 1,3-benzodi-
thiole-2-thione, which can be quantified by high per-
formance liquid chromatography (HPLC) with UV
detection.1–3) This method alone cannot specify the type
of ITC quantified, because 1,3-benzodithiole-2-thione
formation is dependent on the N=C=S moiety but not
the ITC side chain. In individual cases, sulforaphane
(4-methylsulfinylbutyl ITC, SFN) has been detected in
broccoli by gas chromatography/mass spectrometry.4)
SFN and its mercapturic acid pathway conjugates have
been identified and quantified in human urine by HPLC-
Preparation and characterization of ITC-Lys monoclonal antibody.
KLH (5 mg/ml), as a carrier protein, was reacted with four separate
aliquots of 10 mM ITC in 65 mM borate buffer (pH 9.0) for 24 h at
37 ꢁC. Each ITC-modified KLH was then dialyzed against phosphate-
buffered saline (PBS) for 3 d at 4 ꢁC, with several exchanges of PBS.
At the same time, ITC-modified BSA was prepared as a positive
control antigen, as described above. The monoclonal antibody was
prepared by fusion of myeloma and spleen cells by the polyethylene
glycol method, as described previously.7) Monoclonal antibodies
B6C9, 6MS3D10, and PE3A10 were obtained against BITC-KLH,
6MSITC-KLH, and PEITC-KLH, and the immunoglobulin types for
these were identified using an Isotyping Kit (Sigma-Aldrich) as IgG1,
IgG1, and IgG2a respectively. An enzyme-linked immunosorbent assay
(ELISA) was done to screen the hybridomas, and competitive ELISA
was used for characterization, as described previously.7)
Preparation of ITC-modified benzoyl-glycyl-lysine. AITC-modified
BGK was prepared as described previously.7) BITC-, 6MSITC-,
6MTITC-, and PEITC-modified BGK were prepared and purified as
follows: ITC was dissolved in dimethyl sulfoxide (DMSO) at a
concentration of 100 mM. BGK (1 mM) was incubated with 10 mM ITC
y
To whom correspondence should be addressed. Fax: +81-792-93-5710; E-mail: yojikato@shse.u-hyogo.ac.jp
Abbreviations: ITCs, isothiocyanates; AITC, allyl isothiocyanate; BITC, benzyl isothiocyanate; PEITC, phenethyl isothiocyanate; 6MSITC, 6-
methylsulfinylhexyl isothiocyanate; 6MTITC, 6-methylthiohexyl isothiocyanate; SFN, sulforaphane; BSA, bovine serum albumin; KLH, keyhole
limpet hemocyanin; BGK, Nꢀ-benzoyl-glycyl-L-lysine; NAC, Nꢀ-acetyl-L-cysteine; DMSO, dimethyl sulfoxide; ELISA, enzyme-linked
immunosorbent assay; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MRM, multiple reaction monitoring; HPLC, high
performance liquid chromatography

Immunochemical Detection of Food-Derived Isothiocyanate
537
Spin 6). The eluate was diluted 20 times with PBS containing 0.05%
Tween-20 (TPBS) or immunoreaction enhancer solution (Can Get
Signal, Toyobo, Osaka, Japan), and then applied to a microtiter plate.
The wells in the microtiter plate had been separately coated with four
antibodies (0.01 mg/ml in PBS) overnight at 4 ꢁC. After washing and
blocking of the plate using 1% Block Ace aqueous solution (Dainihon
Sumitomo Seiyaku, Osaka, Japan) for 1 h at 37 ꢁC, the diluted eluate
was added and captured by immobilized antibodies for 1 h at 37 ꢁC.
After washing, 100 ml of peroxidase-labeled streptavidin at a 1:5,000
dilution in TPBS was added and this was reacted for 1 h at 37 ꢁC. Color
was developed using tetramethylbenzidine (TMB) substrate solution
(KPL, Gaithersburg MD), and terminated by the addition of 1 M
phosphoric acid. The plate was measured at 450 nm with an xMark
microplate spectrophotometer (Bio-Rad).
O
S
N
C S
N
C S
Allyl ITC (AITC)
6-Methylsulfinylhexyl ITC (6MSITC)
S
N
C S
N
C S
Benzyl ITC (BITC)
6-Methylthiohexyl ITC (6MTITC)
N
C S
Phenethyl ITC (PEITC)
Fig. 1. Chemical Structures of Food-Derived Isothiocyanates (ITCs).
for 0–24 h at 37 ꢁC in 90 mM borate buffer (pH 9.0). To isolate the
adducts, ITC-modified BGK was isocratically separated by reversed-
phase HPLC on a Develosil Combi-RP (20 ꢂ 100 mm) column using
0.1% acetic acid/CH3CN as the eluent at a flow rate of 5.0 ml/min.
The major peak was collected, concentrated, and further purified by
HPLC. The structures of the purified ITCs-BGK adducts were
confirmed by liquid chromatography-mass spectrometry (LC-MS)
and 1H-nuclear magnetic resonance (1H-NMR) analysis using the
JNM-AL series AL300 (JEOL, Tokyo) in CD3OD. NMR spectra were
kindly provided by Dr. Yoshichika Kawai (The University of
Tokushima). The spectral data were as follows: BITC-BGK: 1H-
NMR (ppm) 1.44 (m, 2H), 1.58 (m, 2H), 1.74 (m, 1H), 1.92 (m, 1H),
3.47 (m, 2H), 4.07 (m, 2H), 4.45 (m, 1H), 4.68 (s, 2H), 7.27 (m, 1H),
7.27 (m, 2H), 7.29 (m, 2H), 7.44 (t, J ¼ 7:7, 2H), 7.53 (t, J ¼ 7:5, 1H),
7.84 (d, J ¼ 7:1, 2H); LC-MS (ESIþ) m=z 457.0 ½M þ Hꢃþ. 6MSITC-
BGK: 1H-NMR (ppm) 1.37 (m, 2H), 1.46 (m, 2H), 1.49 (m, 2H), 1.57
(m, 2H), 1.57 (m, 2H), 1.74 (m, 1H), 1.74 (m, 2H), 1.91 (m, 1H), 2.61
(s, 3H), 2.78 (m, 2H), 3.43 (m, 2H), 3.43 (m, 2H), 4.08 (m, 2H), 4.44
(m, 1H), 7.47 (t, J ¼ 7:5, 2H), 7.55 (t, J ¼ 7:3, 1H), 7.87 (d, J ¼ 6:8,
2H); LC-MS (ESIþ) m=z 513.0 ½M þ Hꢃþ. 6MTITC-BGK: 1H-NMR
(ppm) 1.33 (m, 2H), 1.43 (m, 2H), 1.45 (m, 2H), 1.58 (m, 2H), 1.58 (m,
2H), 1.74 (m, 1H), 1.74 (m, 2H), 1.91 (m, 2H), 2.46 (t, J ¼ 7:2, 2H),
2.65 (s, 3H), 3.43 (m, 2H), 3.43 (m, 2H), 4.08 (m, 2H), 4.42 (m, 1H),
7.46 (t, J ¼ 7:5, 2H), 7.54 (t, J ¼ 7:3, 1H), 7.87 (d, J ¼ 7:0, 2H);
LC-MS (ESIþ) m=z 497.0 ½M þ Hꢃþ. In addition, PEITC-BGK was
identified by LC-MS (ESIþ) m=z 471.0 ½M þ Hꢃþ.
Identification of ITCs in shredded wasabi and ground papaya seed
by LC-MS/MS. Suspensions of shredded wasabi and of ground papaya
seed (1.56 to 100 mg/ml) were prepared as described above and
incubated with BGK (1 mM) in water for 24 h at 37 ꢁC. The reaction
mixture was centrifuged (14;000 g, 5 min, 4 ꢁC), and the supernatants
were collected. The sample was analyzed by LC-MS using a quadruple
tandem mass spectrometer (API-3000, Applied Biosystems) connected
to an Agilent 1100 HPLC system. HPLC separation utilized a gradient
system of solvent A (0.1% acetic acid) and solvent B (CH3CN) on a
Develosil ODS-HG-3 (2:0 ꢂ 50 mm) column at a flow rate of 0.2
ml/min. The gradient program was as follows: 0 min (A 100%), 7 min
(A 30%), 7.5 min (A 30%), 7.6 min (A 100%), and 15 min (A 100%).
Quantification of AITC-BGK and BITC-BGK was performed by
multiple reaction monitoring (MRM) of 407.0/308.0 ½M þ Hꢃþ and
457.0/308.0 ½M þ Hꢃþ respectively. A parameter for the detection of
PEITC-BGK (471.0/308.0 ½M þ Hꢃþ) was also included in the
scanning program.
Results
Specificity of monoclonal antibodies to ITC-modified
protein
BITC, 6MSITC, and PEITC (10 mM) were separately
reacted with KLH in borate buffer (pH 9.0) for 24 h, and
the modified KLHs obtained were used as immunogens.
Spleen cells from an immunized mouse and myeloma
cells were fused by the polyethylene glycol method.
After repeated screening and cloning of hybridomas,
three novel monoclonal antibodies, B6C9 (BITC),
6MS3D10 (6MSITC), and PE3A10 (PEITC), were
obtained. The immunoglobulin types of the antibodies
were identified as IgG. None of the antibodies recog-
nized native BSA on noncompetitive indirect ELISA
(data not shown).
Competitive ELISA was used to characterize the
cross-reactivity of the antibodies with the ITC-related
compounds. We have reported that the A4C7 antibody
specifically recognized AITC-modified BGK but not
native BGK (a Lys derivative), allylamine (an analog of
AITC), or AITC-modified NAC.7) In this study, the
antibody also did not react with BITC-BGK or 6MSITC-
BGK (Fig. 2Aa). The B6C9 antibody, which was
prepared by immunization of BITC-modified KLH,
recognized BITC-BGK but not BGK, AITC-BGK, or
6MSITC-BGK (Fig. 2Ab). The 6MS3D10 antibody
recognized 6MSITC-BGK but not BGK, AITC-BGK,
or BITC-BGK (Fig. 2Ac). Although the B6C9 antibody
did not react with BITC-Cys or benzoic acid, it
recognized PEITC-BGK at approximately 10-times
lower levels than BITC-BGK (Fig. 2Ba). The
6MS3D10 antibody recognized 6MTITC-BGK and
6MSITC-NAC at approximately 100-times and 20-times
lower levels than that against 6MSITC-BGK, respec-
Preparation of 6MSITC-modified N-acetyl-cysteine. 6MSITC-NAC
adduct was prepared and purified in a manner similar to that described
previously for AITC-NAC, N-acetyl-S-(N-allylthiocarbamoyl)-L-cys-
teine.7) A NAC solution (10.9 mg/ml in 50% ethanol) was adjusted to
pH 7.8 using 1 M NaOH, and 0.6 ml of 6MSITC solution (13.7 mg/ml
in ethanol) was added to 1.2 ml of NAC solution. The mixture was
stirred under N2 for 3 h at room temperature. The sample was dried by
evaporation and then purified by HPLC.
The major peak was collected using a Develosil Combi-RP
(20 ꢂ 100 mm) column with 0.1% acetic acid/CH3CN (9/1) at a flow
rate of 5.0 ml/min. The purified 6MSITC-NAC adduct was identified
by 1H-NMR and LC-MS. Spectral data were as follows: 1H-NMR
(ppm) 1.37 (m, 2H), 1.46 (m, 2H), 1.61 (m, 2H), 1.7 (m, 2H), 1.89
(s, 3H), 2.56 (s, 3H), 2.75 (m, 2H), 3.45 (d, J ¼ 13:8, 1H), 3.61
(t, J ¼ 6:87, 2H), 3.87 (d, J ¼ 13:8, 1H), 4.57 (m, 1H); LC-MS (ESIþ)
m=z 369.0 ½M þ Hꢃþ.
Immunochemical identification of ITCs in foods by capture of
modified biotin-BSA. Commercial wasabi rhizome was shredded with a
grater, and papaya seed was ground with a mortar and pestle. These
samples were suspended and diluted in pure water to achieve
concentrations of homogenates ranging from 1.56 to 100 mg/ml. As
a standard, authentic AITC and BITC were dissolved in DMSO to a
concentration of 1 M, and then diluted in 0.1 M phosphate buffer
(pH 7.4) to achieve concentrations of AITC and BITC ranging from
0.1 to 10 mM and 0.01 to 1 mM respectively. Biotin-BSA (0.5 mg/ml)
was dissolved in 0.1 M phosphate buffer (pH 7.4) and exposed to
various concentrations of homogenates or authentic ITCs for 24 h at
37 ꢁC. The reaction mixture was centrifuged (14;000 g, 5 min, 4 ꢁC),
and the supernatants were collected. The modified proteins in the
supernatants were purified with a spin column (Bio-Rad, Micro Bio-

538
T. NAKAMURA et al.
A
B
a
1.2
b
b
a
1.2
A4C7
B6C9
B6C9
6MS3D10
1.2
1
1
1
1
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
0.01 0.1
1
10 100
1
1000
0.1
1
10 100 1000
0.01 0.1
1
10 100
0.1
10 100
Competitor conc. (nM)
Competitor conc. (µM)
Competitor conc. (µM)
Competitor conc. (nM)
c
6MS3D10
AITC-BGK
BITC-BGK
6MSITC-BGK
BGK
Benzoic acid
BITC-Cys
PEITC-BGK
6MTITC-BGK
6MSITC-NAC
C
PE3A10
1.2
1.2
1
1
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
0.01 0.1
1
10 100
0.1
1
10
100 1000
Competitor conc. (µM)
Competitor conc. (nM)
D
O
O
COOH
S
O
COOH
S
O
COOH
S
O
S
H
N
H
N
H
N
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
O
O
O
O
(
) AITC-BGK
(
) BITC-BGK
(
) 6MSITC-BGK
S
COOH
O
COOH
S
H
N
H
H2N
N
COOH
S
N
N
NH2
N
N
H
N
H
H
H
H
COOH
O
(
) BGK
(
) Benzoic acid
(
) BITC-Cys
(
) PEITC-BGK
S
O
S
O
COOH
S
H
N
H
N
S
S
N
N
H
N
H
N
H
H
O
COOH
O
(
) 6MTITC-BGK
(
) 6MSITC-NAC
Fig. 2. Characterization of Monoclonal Antibodies by Competitive ELISA.
A, Cross-reactivity of the A4C7 (a), B6C9 (b), and 6MS3D10 (c) antibodies with ITC-BGK (ITC-benzoyl-glycyl-lysine) and BGK as
competitors as determined by competitive ELISA. B, Competitive ELISA with the B6C9 (a) and 6MS3D10 (b) antibodies. C, Competitive
ELISA of the PE3A10 antibody. The cross-reactivity of the antibody with the competitors was expressed as B=B0, in which B is the amount of
the antibody bound to the coating antigen in the presence of the competitor and B0 is the amount in the absence of the competitor. D, Chemical
structures of the competitors used. The symbols used are ( ) AITC-BGK, ( ) BITC-BGK, ( ) 6MSITC-BGK, ( ) BGK, ( ) benzoic acid
(an analog of BITC), ( ) BITC-NAC, ( ) PEITC-BGK (an analog of BITC-BGK), ( ) 6MTITC-BGK (an analog of 6MSITC-BGK), and
(
) 6MSITC-NAC.
tively (Fig. 2Bb). The PE3A10 antibody to PEITC-KLH
reacted with BITC-BGK as well as PEITC-BGK
(Fig. 2C), indicating that the PE3A10 and B6C9 anti-
bodies have similar reactivity. The detection limits of
A4C7, B6C9, 6MS3D10, and PE3A10 were approxi-
mately 1 mM, 1 nM, 0.1 mM, and 10 nM respectively. The
chemical structures of the competitors used are shown in
Fig. 2D.
body but not by the other monoclonal antibodies
(Fig. 3C). Although 6MSITC is one of the characteristic
compounds in wasabi, the 6MS3D10 antibody did not
react with the sample, probably because of the smaller
amount of 6MSITC than AITC in wasabi.8) To improve
sensitivity, binding between the immobilized antibody
and ITC-modified BSA was enhanced by replacing
conventional TPBS with an immunoreaction enhancer
solution. As shown in Fig. 3C (inset), 6MSITC mod-
ification was also confirmed in the wasabi sample. The
product of ground papaya seed incubated with biotin-
BSA reacted with both B6C9 and PE3A10 antibodies,
which have overlapping epitope specificity (Fig. 2).
These results suggest that food-derived ITC can be
identified and semi-quantified by novel antibodies by the
biotin tagged method. Indeed, by replacing biotin-BSA
with a lysine peptide, BGK, the specific formation of
AITC-Lys (from wasabi) and BITC-Lys (from papaya
seed) could be confirmed by LC-MS/MS (Fig. 3D). In
addition, PEITC-BGK was not detected in the reaction
mixture of papaya seed with BGK.
Immunochemical determination of ITCs in foods
We developed a biotin tagged ITC-detection method
by capture ELISA (Fig. 3A). This method specifically
detects ITC-modification of a target compound (BSA).
To verify the method, commercial AITC and BITC were
incubated with biotin-BSA, and the conjugate formed
was analyzed (Fig. 3B). Reactivity increased with
increasing AITC or BITC concentration.
Using this method, we attempted to identify food-
derived ITCs. Suspensions of shredded wasabi and of
ground papaya seed were incubated with biotin-labeled
BSA, and then capture ELISA carried out. The wasabi
sample was specifically recognized by the A4C7 anti-

Immunochemical Detection of Food-Derived Isothiocyanate
539
A
BITC-containing
Biotin
Peroxidase
food
BITC
BITC
Streptavidin
Biotin
BSA
Biotin-BSA
Biotin
BITC
BSA
BSA
ITC-Lys
Reaction of ITC with biotin-BSA
BITC
A4C7
B6C9 6MS3D10 PE3A10
A4C7
B6C9 6MS3D10 PE3A10
Coating the antibody on a microplate
B
C
AITC
BITC
Wasabi
Papaya seed
2
1.5
1
1.6
1.4
1.2
1
1.2
1
1.5
1
0.35
0.3
0.2
0.1
0
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
0
25
50
0.5
0
0.5
0
1
10
100
100
Wasabi conc. (mg/ml) Pa1paya seed conc. (mg/ml)
10
0.01
0.1
1
10
0.1 AITC co1nc. (mM)
BITC conc. (mM)
Coating antibody
Coating antibody
A4C7
B6C9
A4C7
B6C9
6MS3D10
PE3A10
6MS3D10
PE3A10
D
O
COOH
O
COOH
S
H
H
N
R
N C S
N
R
N
NH2
N
H
NH
NH
LC-MS/MS
H
ITC
O
O
Food-derived ITC
BGK
ITC-modified BGK
Wasabi+BGK
Papaya+BGK
BITC-BGK
AITC-BGK
[M+H]+ 457.0/308.0
[M+H]+ 407.0/308.0
12.5 mg/ml
1.56 mg/ml
0 mg/ml
12.5 mg/ml
1.56 mg/ml
0 mg/ml
8.0
9.0
10.0
9.0
10.0
Time (min)
11.0
Time (min)
Fig. 3. Capture Assay of Food-Derived ITC Using Biotin-BSA as Carrier Protein.
A, Scheme for the reaction of immobilized antibody on a microplate with ITC-modified biotin-BSA as an illustration using a BITC-containing
food. ITC in food reacts with biotin-BSA at neutral pH. The modified BSA was captured by immobilized antibody. The binding was evaluated by
reaction with peroxidase-labeled streptavidin. B, Confirmation of the assay using pure ITCs. Biotin-BSA (0.5 mg/ml) was exposed to authentic
AITC (0.1 to 10 mM) or BITC (0.01 to 1 mM) for 24 h at 37 ꢁC. Symbols: ( ) the A4C7 antibody, ( ) the B6C9 antibody, ( ) the 6MS3D10
antibody, ( ) the PE3A10 antibody. C, The reactivity of the antibodies with food-derived ITC-modified biotin-BSA was measured. Shredded
wasabi and ground papaya seed (1.56 to 100 mg/ml) were suspended individually in water and then incubated with biotin-BSA (0.5 mg/ml) for
24 h at 37 ꢁC. The symbols are the same as in B. Control and wasabi samples (0, 25, 50 mg/ml) were also reacted with the 6MS3D10 antibody in
an immunoreaction enhancer solution instead of TPBS. Then the binding of biotin-BSA to the antibody was evaluated by reaction with
peroxidase-labeled streptavidin (C, inset in wasabi). D, Shredded wasabi and groþund papaya seed (1.56–100 mg/ml) were reacted with BGK
(1 mM), as a model Lys residue, for 24 h at 37 ꢁC. The MRM 407.0/308.0 ½M þ Hꢃ for AITC-BGK and 457.0/308.0 ½M þ Hꢃþ for BITC-BGK
were scanned by LC-MS/MS.
Discussion
(Fig. 1). Similarly, the 6MS3D10 antibody recognized
the 6MTITC-Lys adduct to some extent. This antibody
reacted with Cys conjugate, a predominant ITC con-
jugate.9) Although Cys conjugate is known to be
unstable,10,11) covalent modification of ITC with Cys
from ꢀ-tubulin in cells has been identified by mass
spectrometry.12) The 6MS3D10 antibody might be
useful for detection of ITC-Cys adducts in cell and
tissue samples if the adducts are stably isolated.
When testing our antibodies with food-derived
ITC-biotin-BSA conjugates, we saw that A4C7 and
6MS3D10 reacted with the reaction mixture of shredded
wasabi and biotin-BSA (Fig. 3C). Even though 6MSITC
In the present study, we developed three novel
monoclonal antibodies against BITC, 6MSITC, and
PEITC-modified protein. All these antibodies recog-
nized the corresponding ITC-modified BSA (data not
shown), and peptides that contained ITC-modified Lys.
Although we also obtained a monoclonal antibody
reactive to SFN-modified protein, the antibody did not
react with free SFN-Lys derivative (data not shown).
The B6C9 and PE3A10 antibodies exhibited cross-
reactivity towards BITC and PEITC (Fig. 2), probably
due to the similar structures of these Lys adducts

540
T. NAKAMURA et al.
is present in amounts approximately 6.5-times less than
AITC in wasabi,8) we were able to detect 6MSITC. We
established a brief and convenient assay for the
detection of ITCs in food homogenates. BITC-Lys was
detected by the B6C9 antibody in the mixture of ground
papaya seed and biotin-BSA (Fig. 3C). While the
PE3A10 antibody, prepared from PEITC-KLH as the
immunogen, reacted with this mixture, PEITC was not
present in the papaya seed, and this was due to cross-
reaction between antibody B6C9 and PE3A10.
With regard to sensitivity and selectivity, chemical
analysis by LC-MS/MS is generally superior to immu-
nochemical methods. However, as compared with LC-
MS/MS, our immunochemical assay can handle many
samples at once. The sensitivity of the immunochemical
assay was increased by the use of immunoreaction
enhancer solution. It has been reported that accumula-
tion of quercetin metabolite in human atherosclerotic
lesions was immunohistochemically proven by a mono-
clonal antibody specific to the quercetin metabolite.13) In
this way, an immunochemical technique is a powerful
tool not only for identifying molecules in foods, but also
for evaluating the localization of target molecules in
tissues and in cellular components. The antibodies
obtained might be useful tools for the identification of
ITC in these applications.
Dr. Yoshichika Kawai (The University of Tokushima)
for helpful technical assistance, and Dr. Yoshimasa
Nakamura (Okayama University) for helpful advice.
References
1) Zhang Y, Cho CG, Posner GH, and Talalay P, Anal. Biochem.,
205, 100–107 (1992).
2) Zhang Y, Wade KL, Prestera T, and Talalay P, Anal. Biochem.,
239, 160–167 (1996).
3) Zhang Y and Talalay P, Cancer Res., 58, 4632–4639 (1998).
4) Chiang WCK, Pusateri DJ, and Leitz REA, J. Agric. Food
Chem., 46, 1018–1021 (1998).
5) Egner PA, Kensler TW, Chen JG, Gange SJ, Groopman JD,
and Friesen MD, Chem. Res. Toxicol., 21, 1991–1996
(2008).
6) Yamaguchi H, Noshita T, Kidachi Y, Umetsu H, Fuke Y, and
Ryoyama K, Chem. Pharm. Bull., 56, 715–719 (2008).
7) Nakamura T, Kawai Y, Kitamoto N, Osawa T, and Kato Y,
Chem. Res. Toxicol., 22, 536–542 (2009).
8) Fukuchi Y, Kato Y, Okunishi I, Matsutani Y, Osawa T, and
Naito M, Food Sci. Technol. Res., 10, 290–295 (2004).
9) Conaway CC, Krzeminski J, Amin S, and Chung FL, Chem.
Res. Toxical., 14, 1170–1176 (2001).
10) Hong F, Freeman ML, and Liebler DC, Chem. Res. Toxicol., 18,
1917–1926 (2005).
11) Kassahun K, Davis M, Hu P, Martin B, and Baillie T, Chem.
Res. Toxicol., 10, 1228–1233 (1997).
12) Mi L, Xiao Z, Hood BL, Dakshanamurthy S, Wang X, Govind
S, Conrads TP, Veenstra TD, and Chung FL, J. Biol. Chem.,
283, 22136–22146 (2008).
Acknowledgments
13) Kawai Y, Nishikawa T, Shiba Y, Saito S, Murota K, Shibata N,
Kobayashi M, Kanayama M, Uchida K, and Terao J, J. Biol.
Chem., 283, 9424–9434 (2008).
We thank Kinjirushi Co., Ltd. (Nagoya, Japan) for
kindly providing 6MSITC and 6MTITC. We thank