Biochemical and Biophysical Research Communications 406 (2011) 608–613

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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Establishment of a novel monoclonal antibody SMab-1 speciﬁc for IDH1-R132S
mutationMika Kato Kaneko a, Wei Tian a, Shingo Takano b, Hiroyuki Suzuki c, Yoshihiko Sawa d, Yasukazu Hozumi e,
Kaoru Goto e, Kentaro Yamazaki f, Chifumi Kitanaka g, Yukinari Kato a,⇑a
Molecular Tumor Marker Research Team, The Oncology Research Center, Advanced Molecular Epidemiology Research Institute, Yamagata University Faculty of Medicine, 2-2-2
Iida-nishi, Yamagata 990-9585, Japanb
Department of Neurosurgery, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8575, Japanc
Department of Experimental Pathology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8575, Japand
Section of Functional Structure, Department of Morphological Biology, Division of Biomedical Sciences, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japane
Department of Anatomy and Cell Biology, Yamagata University School of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585, Japanf
Department of Forensic Medicine, Yamagata University School of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585, Japang
Department of Molecular Cancer Science, Yamagata University School of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585, Japana r t i c l e

i n f oArticle history:
Received 17 February 2011
Available online 23 February 2011
Keywords:
IDH1
Mutation
R132S
Monoclonal antibody
Glioblastomaa b s t r a c t
Isocitrate dehydrogenase 1 (IDH1) mutations, which are early and frequent genetic alterations in gliomas,
are speciﬁc to a single codon in the conserved and functionally important Arginine 132 (R132) in IDH1.
We earlier established a monoclonal antibody (mAb), IMab-1, which is speciﬁc for R132H-containing
IDH1 (IDH1-R132H), the most frequent IDH1 mutation in gliomas. To establish IDH1-R132S-speciﬁc
mAb, we immunized mice with R132S-containing IDH1 (IDH1-R132S) peptide. After cell fusion using
Sendai virus envelope, IDH1-R132S-speciﬁc mAbs were screened in ELISA. One mAb, SMab-1, reactedwith the IDH1-R132S peptide, but not with other IDH1 mutants. Western-blot analysis showed that
SMab-1 reacted only with the IDH1-R132S protein, not with IDH1-WT protein or IDH1 mutants, indicating that SMab-1 is IDH1-R132S-speciﬁc. Furthermore, SMab-1 speciﬁcally stained the IDH1-R132Sexpressing glioblastoma cells in immunocytochemistry and immunohistochemistry, but did not reactwith IDH1-WT or IDH1-R132H-containing glioblastoma cells. We newly established an anti-IDH1R132S-speciﬁc mAb SMab-1 for use in diagnosis of mutation-bearing gliomas.
Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction
Isocitrate dehydrogenase 1 (IDH1) mutations, which have been
identiﬁed as early and frequent genetic alterations (50–93%) in
astrocytomas, oligodendrogliomas, and oligoastrocytomas, as wellas in secondary glioblastomas, might be the initiating event inthese glioma subtypes [1–4]. In contrast, primary glioblastomas,
as well as other systemic cancers, rarely contain IDH1 mutations.
The IDH1 mutations are remarkably speciﬁc to a single codon in
the conserved and functionally important Arginine 132 residue(R132) in IDH1. Mutations in the analogous IDH2-R172 codon also
occur at a lower rate (3–5%) in these cancers. Of interest, IDH1/2
mutations were observed subsequently in 22% of acute myeloid
leukemias [5]. IDH1 mutations were found to result in the abilityof the enzyme to catalyze the reduced NADP-dependent reduction

Abbreviations: IDH1, isocitrate dehydrogenase 1; mAb, monoclonal antibody.

⇑ Corresponding author. Fax: +81 23 628 5295.

E-mail address: yukinari-k@bea.hi-ho.ne.jp (Y. Kato).
0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2011.02.102

of a-ketoglutarate to R()-2-hydroxyglutarate (2-HG) [6]. Reduction of a-ketoglutarate by 2-HG or mutant IDH1 results in a lower
level of prolyl hydroxylases and promotes the accumulation of hypoxia-inducible factor (HIF) 1a [7]. HIF-1a levels were greater inhuman gliomas harboring an IDH1 mutation than in tumors without the mutation. Therefore, IDH1 appears to function as a tumor
suppressor that, when mutationally inactivated, contributes totumorigenesis in part through induction of the HIF-1 pathway.
To date, two monoclonal antibodies against IDH1 mutations
have been reported [8,9]. Of those antibodies, we establishedIMab-1, which is speciﬁc for IDH1-R132H, the most frequent
IDH1 mutations (>90%) [8]. IMab-1 reacted with the IDH1-R132H
peptide, but not with the wild type IDH1 (IDH-WT) peptide in ELISA. In Western-blot analysis, IMab-1 reacted only with the IDH1R132H protein, not the IDH1-WT protein or other IDH1 mutants.
Recently, we investigated the usefulness of IMab-1 for immunohistochemical analysis of numerous human glioma samples in comparison with the direct DNA sequencing method [10]. IMab-1
detected 12 out of 49 cases. However, only nine cases were deter-M.K. Kaneko et al. / Biochemical and Biophysical Research Communications 406 (2011) 608–613

mined to be IDH1-R132H by direct DNA sequencing because of thesmall population of IDH1-R132H mutation-possessing tumor cells,
indicating that IMab-1 immunohistochemistry is useful for detecting IDH1-R132H in immunohistochemistry.
Although several IDH1 mutations exist, including R132H,
R132C, R132S, R132G, and R132L, IMab-1 and another mAb, H09,
can detect only IDH1-R132H mutation [11]. Therefore, novel antibodies that recognize the other IDH1 mutants should be developed
to cover all IDH1 mutations in immunohistochemistry. We newly
report an anti-IDH1-R132S-speciﬁc monoclonal antibody SMab-1,
which is expected to be extremely useful for diagnosis of mutation-bearing gliomas and acute myeloid leukemia.
2. Materials and methods
2.1. Animals, cell lines, and tissues
Female BALB/c mice were obtained from CLEA Japan Inc. (Tokyo,
Japan). P3U1 and LN229 cells were obtained from the American
Type Culture Collection (ATCC, Manassas, VA). P3U1 or LN229 cells
were cultured at 37 °C in a humidiﬁed atmosphere of 5% CO2 and
95% air in RPMI 1640 or DMEM medium (Wako Pure Chemical
Industries Ltd., Osaka, Japan), respectively, including 2 mM L-glutamine (Wako Pure Chemical Industries Ltd.) supplemented with
10% heat-inactivated fetal bovine serum (FBS; Invitrogen Corp.,
Carlsbad, CA). Three glioblastoma patients who underwent primary surgery between 2009 and April 2010 at Tsukuba University
Hospital were included in this study [10]. Informed consent was
obtained from each patient or the patient’s guardian for obtaining
samples and for subsequent data analysis.
2.2. Hybridoma production
BALB/c mice were immunized by i.p. injections of 100 lg of synthetic peptide CKPIIIGSHAYGD (IDH1-R132S peptide; Operon Biotechnologies, K.K., Tokyo, Japan), corresponding to amino acids
126–137 of human IDH1-R132S plus N-terminus cysteine conjugated with KLH together with Imject Alum (Thermo ScientiﬁcInc., Rockford, IL). One week later, secondary i.p. immunization of
30 lg of IDH1-R132S peptide was performed. After several additional immunization of 30 lg of IDH1-R132S peptide, a booster
injection was given i.p. 2 days before spleen cells were harvested.
The spleen cells were fused with mouse myeloma P3U1 cells using
Sendai virus (Hemagglutinating Virus of Japan: HVJ) envelope:
GenomONE-CF (Ishihara Sangyo Kaisha Ltd., Osaka, Japan) according to the manufacturer’s instructions [8,12]. The hybridomas weregrown in RPMI medium with hypoxanthine, aminopterin, and thymidine selection medium supplement (Invitrogen Corp.). The culture supernatants were screened using ELISA for binding to the
IDH1-R132S peptide and the IDH1-wild type (IDH1-WT), as described in Section 2.3.
2.3. Enzyme-linked immunosorbent assay (ELISA)
Synthetic peptides (Operon Biotechnologies, K.K.) corresponding
to amino acids 126–137 of the human IDH1 plus N-terminus cysteine: IDH1-WT: (CKPIIIGRHAYGD), IDH1-R132H (CKPIIIGHHAYGD), IDH1-R132C (CKPIIIGCHAYGD), IDH1-R132S (CKPIIIGSHAYGD), IDH1-R132G (CKPIIIGGHAYGD), and IDH1-R132l (CKPIIIGLHAYGD) were immobilized, respectively, on Maxisorp 96-wellimmunoplates (Thermo Fisher Scientiﬁc Inc., Waltham, MA) at
1 lg/ml for 30 min, respectively. After blocking with 1% BSA in
PBS, the plates were incubated with culture supernatant, followedby 1:1000 diluted peroxidase-conjugated anti-mouse IgG (Dako,
Glostrup, Denmark). The enzymatic reaction was conducted with a609

substrate solution containing TMB (Thermo Fisher Scientiﬁc Inc.).
After the reaction was stopped with 2 M H2SO4, the optical density
was measured at 450 nm with a Benchmark microplate reader(Bio-Rad Laboratories Inc., Philadelphia, PA). These reactions were
performed with a volume of 50 ll at 37 °C.

2.4. Plasmid preparation
Human IDH1 cDNA (GenBank Accession No. AF113917 or
BC012846) encoding a full-length open reading frame (ORF) was
obtained by PCR using a human lung cDNA library (Cosmo Bio
Co. Ltd., Tokyo, Japan) as a template. The primer set for IDH1 wasasfollows:
EcoRI-IDH1-F1,
50 -cacgaattcATGTCCAAAAAAA00TCAGTGG-3 and SalI-IDH1-R1, 5 -gtggtcgacTTAAAGTTTGGCCTG
AGCTA-30 . The ampliﬁed cDNA was subcloned into a pcDNA3.1/
V5-His-TOPO vector (Invitrogen Corp.). Substitution of the Arginine 132 (R132) to appropriate amino acid codons (Histidine, Cysteine, or Serine) in IDH1 cDNAs was accomplished using the
QuikChange Lightning site-directed mutagenesis kit (Stratagene,
La Jolla, CA). The full-length IDH1-WT, R132H, R132C, and R132S
were subcloned into an expression vector, pMAL-c2 (New England
Biolabs Inc., Beverly, MA), via EcoRI and SalI restriction sites.

2.5. Protein expression using bacteria cells and mammalian cells
Competent Escherichia coli TOP-10 cells (Invitrogen Corp.) weretransformed with the plasmid, pMAL-IDH1-WT, -R132H, -R132C,
and -R132S. Then they were cultured overnight at 37 °C in LB medium containing 100 lg/ml ampicillin. Cell pellets were resuspended in phosphate buffered saline (PBS) with 1% Triton X-100
with 50 lg/ml aprotinin. After sonication, the crude extracts werecollected by centrifugation (9000g for 30 min, 4 °C). The supernatants were loaded onto Amylose resin. The loaded resins were
washed extensively with column buffer (20 mM Tris–HCl, pH 7.4,
200 mM NaCl, 1 mM EDTA), and the fusion proteins were elutedby column buffer with 10 mM maltose. Mammalian cells weretransfected with appropriate amounts of plasmids, pcDNA3.1/
IDH1-R132H or -R132S using Metafectene (Biontex Laboratories
GmbH, Martinsried, Germany) according to the manufacturers’
instructions. Stable transfectants were selected by cultivating thetransfectants in medium containing 0.5 mg/ml of Geneticin (Invitrogen Corp.). The expression level of IDH1 was conﬁrmed using
Western-blot analysis.

2.6. Western-blot analyses
Cultured cell pellets were lysed with PBS with 1% Triton X-100
for 30 min on ice. The lysate supernatants were centrifuged for
15 min at 15,000 rpm to remove cellular debris. Cell lysates containing 5 lg of total protein were prepared for Western-blot analysis by boiling in SDS sample buffer (50 mM Tris, 2% SDS, 5%
glycerol, 10% 2-mercaptoethanol, pH 6.8). They were electrophoresed on 5–20% polyacrylamide gels (Wako Pure Chemical Industries
Ltd., Osaka, Japan). The separated proteins were transferred to a
PVDF membrane (Bio-Rad Laboratories Inc.). After blocking with
4% skim milk in PBS, the membrane was incubated with antiIDH1 antibody (1/1000 diluted; Santa Cruz Biotechnology Inc., Santa Cruz, California), IMab-1 culture supernatant (kindly donated by
Dr. Darell D. Bigner, Duke University Medical Center), or SMab-1
culture supernatant; then with peroxidase-conjugated secondary
antibodies (1/1000 diluted) and developed with ECL reagents (GEHealthcare, Buckinghamshire, UK) using EZ-capture II (Atto Corp.,
Tokyo, Japan).

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M.K. Kaneko et al. / Biochemical and Biophysical Research Communications 406 (2011) 608–613

2.7. Immunocytochemical analysis
Cultured cells were ﬁxed with 4% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.4) for 20 min at room temperature andthen permeabilized with 0.1% Triton X-100 in PBS for 15 min. Cells
were then treated with 10% normal goat serum in PBS (NGS/PBS) to
block nonspeciﬁc binding sites and were incubated with SMab-1 orIMab-1 culture supernatant containing 0.1% Triton X-100 overnight at 4 °C in a moist chamber. Then they were incubated withgoat anti-mouse IgG-Alexa 488 (dilution 1:400; Molecular Probes
Inc., Eugene, OR) in PBS containing 0.1% Triton X-100 for 1 h atroom temperature. Cells were also treated with TO-PRO-3 (Molecular Probes) to stain the cell nuclei. They were examined using
confocal laser-scanning microscopy (LSM5 PASCAL; Carl Zeiss
Inc., Jena, Germany). Then ﬂuorescent images were processedusing image processing software (Adobe Photoshop; Adobe Systems Inc.).
2.8. Immunohistochemical analysis
IDH1-R132S protein expression was determined immunohistochemically in parafﬁn-embedded tumor specimens, as described
previously [10]. Brieﬂy, 5-lm-thick histologic sections were deparafﬁnized in xylene and rehydrated. Then they were heated at
100 °C in citrate buffer (pH 6.0) for 5 min. Sections were incubatedwith culture supernatants of SMab-1 or IMab-1 overnight at 4 °C.
The LSAB Kit (Dako, Glostrup, Denmark) was used for the post-primary antibody blocker and secondary antibody. Color was developed using 3, 3-diaminobenzidine tetrahydrochloride (DAB) for
10 min, and counterstained with hematoxylin.
3. Results and discussion
3.1. Production of an IDH1-R132S-speciﬁc antibody
The IDH1 gene at 2q33 encodes isocitrate dehydrogenase 1(IDH1), which catalyzes the oxidative carboxylation of isocitrate
to a-ketoglutarate [7]. IDH1 mutations occur frequently in somemalignant gliomas [2] and less frequently in acute myeloid leukemias [5]. To date, almost all IDH1 mutations have been identiﬁed at
the critical isocitrate binding site R132 codon. The vast majority ofchanges are heterozygous [1–4]. In gliomas, mutations in the IDH1
homolog IDH2 have been identiﬁed at the aligned R172 codon [2].
These discoveries mark unique ﬁndings in the ﬁeld of cancer
genetics.
We previously established IMab-1, which is speciﬁc for IDH1R132H, the most frequent of IDH1 mutations (>90%) [8]. However,antibodies against other IDH1 mutations have not been reported.
For this study, we immunized mice with synthetic peptides of
IDH1-R132S mutant. After cell fusion using the Sendai virus envelope, the wells of hybridomas, which produced IDH1-R132S-speciﬁc antibodies, were selected in ELISA. In total, 46 wells, which
reacted with IDH1-R132S, not IDH1-WT, were selected. However,
45/46 wells did not react with IDH1-R132S protein in ELISA. After
limiting dilution, one clone, SMab-1 (IgG1 subclass), wasestablished.

with IDH1-R132H peptide, not with IDH1-WT or other IDH1 mutants (R132C, R132S, R132G, R132L) peptides (data not shown),
indicating that SMab-1 is a speciﬁc antibody against IDH1-R132S
peptide. To conﬁrm the reactivity of SMab-1 antibody against not
only IDH1 peptides but also against IDH1 proteins, IDH1-WT andthree IDH1 mutants (R132H, R132C, R132S) were expressed in
E. coli for Western-blot analyses. As portrayed in Fig. 1A, SMab-1
recognized only IDH1-R132S protein, not the other proteins (WT,R132H, R132C), indicating that SMab-1 is a speciﬁc antibody
against IDH1-R132S protein. IMab-1 recognized only IDH1R132H protein (Fig. 1B); an anti-IDH1 antibody reacted with all
types (Fig. 1C). These results indicate that SMab-1 is also useful
in detecting not only IDH1-R132S peptide, but also the IDH1R132S protein.
3.3. Immunocytochemical and immunohistochemical analyses by
SMab-1 against glioma tissues with IDH1-R132S
SMab-1 was conﬁrmed to be an anti-IDH1-R132S-speciﬁc antibody in ELISA and Western-blot analysis. Therefore, we ﬁrst performed immunocytochemical analyses using IDH1-R132S or
IDH1-R132H-transfected LN229 glioblastoma cells. Results showthat SMab-1 reacted with IDH1-R132S-transfected cells (Fig. 2A),
not with IDH1-R132H-transfected cells (Fig. 2C), whereas IMab-1
reacted with IDH1-R132H-transfectant cells (Fig. 2G), but not with
IDH1-R132S-transfected cells (Fig. 2E), indicating that both SMab1 and IMab-1 are mutation-speciﬁc antibodies in immunocytochemistry. We next performed immunohistochemistry of SMab-1
against IDH1-R132S-positive or IDH1-R132H-positive gliomas.
Typical results are shown in Fig. 3. SMab-1 stained almost all tumor cells of IDH1-R132S-positive gliomas (Fig. 3A), although nostaining was observed in IDH1-R132H-positive gliomas or IDH1WT gliomas (Fig. 3C and E). In fact, SMab-1 did not stain endothelial cells in IDH1-R132S-positive gliomas (Fig. 3A). Furthermore,
IMab-1, also used as mouse IgG1 isotype control, did not stain
any tumor cells in IDH1-R132S-positive gliomas (Fig. 3B), whereas
IMab-1 stained IDH1-R132H-positive gliomas (Fig. 3D). These results indicate that SMab-1 is useful in immunohistochemical analyses for detection of IDH1-R132S mutation. Our previous reports
described that one R132S-positive glioblastoma among 13 IDH1mutation-bearing gliomas was undetectable by IMab-1 antibody[10]. We were able to raise the detection ratio of IDH1 mutation

3.2. Speciﬁcity of SMab-1 against IDH1 mutants
To determine the speciﬁcity of SMab-1 monoclonal antibody,the reactivities against IDH1-wild type (IDH1-WT) and the IDH1
mutants (R132H, R132C, R132S, R132G, R132L) peptides were
investigated using ELISA. Results show that SMab-1 reacted with
IDH1-R132S peptide, not with IDH1-WT or other IDH1 mutants(R132H, R132C, R132G, R132L) peptides, whereas IMab-1 reacted

Fig. 1. Western-blot analysis by SMab-1. Cell lysates from E. coli-expressing IDH1wild type (lane 1) and IDH1 mutants (lane 2, R132H; lane 3, R132C; lane 4, R132S)
were electrophoresed under reducing conditions using 5–20% gels, and wereWestern-blotted with SMab-1 (A), IMab-1 (B), and anti-IDH1 (C).

M.K. Kaneko et al. / Biochemical and Biophysical Research Communications 406 (2011) 608–613

611

Fig. 2. Immunocytochemical analysis by SMab-1 against IDH1-R132S-transfected glioblastoma cells. LN229 cells were transfected by IDH1-R132S (A, B, E, and F) or IDH1R132H (C, D, G, and H). Each cell was stained by SMab-1 (A–D) or by IMab-1 (E–H) followed by anti-mouse IgG-Alexa 488 antibody (green). Cells were also treated with TOPRO-3 to stain the cell nuclei (blue) (B, D, F, and H). Bars 50 lm. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version ofthis article.)612

M.K. Kaneko et al. / Biochemical and Biophysical Research Communications 406 (2011) 608–613

Fig. 3. Immunohistochemical analysis by SMab-1 against glioma tissues with IDH1 mutations. Glioblastoma tissues having IDH1-R132S (A and B), IDH1-R132H (C and D), andwild type (E and F) were stained with SMab-1 (A, C, and E) or IMab-1 (B, D, and F). Magniﬁcation: 200.

in immunohistochemistry by using both IMab-1 and SMab-1 forthose glioma samples. IDH1 mutation were reported as very early
events, before TP53 mutations or loss of 1p/19q occurs [13], whichmight engender diffused staining by SMab-1 or IMab-1 in almost
every cell of IDH1-R132S or IDH1-R132H-positive glioma, respectively (Fig. 3A and D).
Recently, IDH1/2 mutations have been found in 129 of 805
acute myeloid leukemia (AML)—IDH1 in 61 patients (7.6%) andIDH2 in 70 patients (8.7%) [14]. It is particularly interesting thatﬁve of 61 IDH1-mutant-bearing patients (8.2%) were R132S,
although only 1.3% of IDH1 mutations were R132S in gliomas[11], indicating that SMab-1 might be more useful for diagnosis
of IDH1-R132S-positive AMLs. The R132C mutations were observed at high frequency in AML (20/61 IDH1 mutations; 33%),
although they were observed in only 3.7% of glioma specimens[11,14], suggesting that an anti-IDH1-R132C speciﬁc antibody
might be useful for diagnosis of AML as well as glioma subtypes.
In conclusion, the established anti-IDH1-R132S-speciﬁc
monoclonal antibody SMab-1 is expected to be extremely usefulfor diagnosis and biological evaluation of mutation-bearing
gliomas in combination with anti-IDH1-R132H-speciﬁc monoclonal antibody IMab-1. Additional IDH1/2 mutation-speciﬁc antibodies must be developed for pathological diagnosis of gliomas andAML.

Acknowledgments
We thank Ryo Yanagiya, Shunpei Morita, and Yoshiko Tsukada
for their excellent technical assistance. This study was supported
in part by grants from the Global COE Program for Medical Sciences, Japan Society for Promotion of Science, from the Ministryof Education, Culture, Sports, Science and Technology of Japan,
and from the Mitsubishi Pharma Research Foundation. T.W. was
supported by Experimental and Research Center, North China Coal
Medical University.
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