Biochemical and Biophysical Research Communications 432 (2013) 564–567

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

A novel monoclonal antibody GMab-m1 speciﬁcally recognizes
IDH1-R132G mutation
Yukinari Kato a,b,⇑, Atsushi Natsume c, Mika Kato Kaneko a,baRegional Innovation Strategy Support Program, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
Molecular Tumor Marker Research Team, Global COE Program, Yamagata University Faculty of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585, Japanc
Department of Neurosurgery, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japanba r t i c l e

i n f oArticle history:
Received 5 February 2013
Available online 26 February 2013
Keywords:
IDH1 mutation
R132G
Monoclonal antibody

a b s t r a c t
Isocitrate dehydrogenase 1 (IDH1) mutations occur in gliomas, acute myeloid leukemias, and cartilaginous tumors. While IDH1 catalyzes the oxidative carboxylation of isocitrate to a-ketoglutarate in cytosol,
mutated IDH1 proteins possess the ability to change a-ketoglutarate into onco-metabolite R(-)-2hydroxyglutarate (2-HG). To data, two monoclonal antibodies (mAbs), which are speciﬁc for IDH1 mutations have been established: clone HMab-1 against IDH1-R132H and clone SMab-1 against IDH1-R132S.
However, speciﬁc mAbs against IDH1-R132G, which are useful for immunohistochemical analysis, havenot been reported. To establish IDH1-R132G-speciﬁc mAbs, we immunized mice with IDH1-R132G-containing peptides. Established mAb GMab-m1 reacted with the IDH1-R132G peptide, but not with IDH1wild type (WT) in ELISA. Western-blot analysis also showed that GMab-m1 reacted with the IDH1-R132G
recombinant proteins, not with IDH1-WT or other IDH1 mutants, indicating that GMab-m1 is IDH1R132G-speciﬁc. Furthermore, GMab-m1 speciﬁcally stained the IDH1-R132G-expressing glioma cells in
immunohistochemistry. This is the ﬁrst report to establish anti-IDH1-R132G-speciﬁc mAbs, which is useful in immunohistochemistry of IDH1-R132G-bearing tumors.
Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction
Isocitrate dehydrogenase 1 (IDH1) mutations were found to result in the ability of the enzyme to change a-ketoglutarate to oncometabolite R(-)-2-hydroxyglutarate (2-HG), although IDH1 usuallycatalyzes the oxidative carboxylation of isocitrate to a-ketoglutarate in cytosol [1]. Excess accumulation of 2-HG leads to an elevatedrisk of malignant brain tumors in patients. IDH1 mutations occur insome gliomas [2], cartilaginous tumors [3,4], and in acute myeloid
leukemias [5]. In astrocytomas, oligodendrogliomas, oligoastrocytomas, and secondary glioblastomas, IDH1 mutations have been
identiﬁed as early and frequent genetic alterations (50–93%), andmight be the initiating event in these glioma subtypes [2,6–8]. In
contrast, primary glioblastomas rarely contain IDH1 mutations.
The IDH1 mutations are remarkably speciﬁc to a single codon in
the conserved and functionally important Arginine 132 residue(R132). The vast majority of changes are heterozygous. IDH1 mutaAbbreviations: IDH1, isocitrate dehydrogenase 1; mAb, monoclonal antibody.

⇑ Corresponding author at: Regional Innovation Strategy Support Program,Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai,
Miyagi 980-8575, Japan. Fax: +81 23 628 5295.
E-mail addresses: yukinari-k@bea.hi-ho.ne.jp, yukinarikato@med.tohoku.ac.jp(Y. Kato).
0006-291X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bbrc.2013.02.049

tions were reported as IDH1-R132H (664/716: 92.7%), IDH1-R132C(29/716: 4.2%), IDH1-R132S (11/716: 1.5%), IDH1-R132G (10/716:
1.4%), and IDH1-R132L (2/716: 0.2%) [9]. Only one IDH1-R132V
mutation was also reported in 1212 IDH1 mutations (1/1212:
0.1%) [10] Although one monoclonal antibody (mAb) against
IDH1-R132H has been reported by another group [11], we established IDH1-R132H-speciﬁc mAb HMab-1 [12] and IDH1-R132Sspeciﬁc mAb SMab-1 [13]. We previously evaluated 164 cases ofglioma for IDH1 mutations (R132H and R132S) using anti-IDH1
mAbs: HMab-1 and SMab-1 [14]. IDH1 mutation was detected in
9.7%, 63.6%, 51.7%, and 77.8% of primary grade IV, secondary gradeIV, grade III, and grade II gliomas, respectively. Evaluation of IDH1
mutation using immunohistochemistry might be of greater prognostic signiﬁcance than histological grading alone in grade III and
grade IV gliomas. Combination of HMab-1 and SMab-1 is able toﬁnd more than 90% of IDH1 mutations of gliomas in immunohistochemistry; however, other IDH1 mutations have been missed in
immunohistochemistry. Therefore, novel antibodies that recognize
other IDH1-mutations should be developed to cover all of IDH1
mutations in immunohistochemistry. Here, we report a novelanti-IDH1-mutation-speciﬁc monoclonal antibody, GMab-m1,
which is expected to be extremely useful for diagnosis of IDH1R132G-bearing gliomas.

Y. Kato et al. / Biochemical and Biophysical Research Communications 432 (2013) 564–567

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2. Materials and methods

2.5. Protein expression using bacteria cells and mammalian cells

2.1. Cell lines and tissues

Competent Escherichia coli TOP-10 cells (Life Technologies
Corp.) were transformed with the plasmid, pMAL-IDH1-WT,
pMAL-IDH1-R132G, and pMAL-IDH1-R132L. Then, they were cultured overnight at 37 °C in LB medium (Life Technologies Corp.)
containing 100 lg/ml ampicillin (Sigma–Aldrich). Cell pellets were
resuspended in phosphate buffered saline (PBS) with 1% Triton X100 with 50 lg/ml aprotinin (Sigma–Aldrich). After sonication
using Branson Advanced Soniﬁer (Branson Ultrasonics, Danbury,CT), the crude extracts were collected by centrifugation (9000  g
30 min, 4 °C). The supernatants were loaded onto Amylose resin(GE Healthcare, Buckinghamshire, UK). 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. CHO cells were transfectedwith appropriate amounts of plasmids, pcDNA3/IDH1-WT,
pcDNA3/IDH1-R132G, and pcDNA3/IDH1-R132L using Lipofectamine LTX (Life Technologies Corp.) according to the manufacturer’s instructions. Stable transfectants were selected bycultivating the transfectants in medium containing 1 mg/ml of
G418 (Wako Pure Chemical Industries Ltd.). The expression levelof IDH1 was conﬁrmed using Western-blot analysis.

Chinese hamster ovary (CHO)-K1 cells (CCL-61) and Sp2/0-Ag14
myeloma (CRL-1581) and were obtained from the American Type
Culture Collection (ATCC, Manassas, VA). These cell lines were cultured at 37 °C in a humidiﬁed atmosphere of 5% CO2 and 95% air in
RPMI 1640 medium including 2 mM L-glutamine (Nakalai Tesque,Inc., Kyoto, Japan) supplemented with 10% heat-inactivated fetal
bovine serum (FBS; Life Technologies Corp., Carlsbad, CA). One glioma patient (oligodendroglioma, WHO grade II), who underwent
primary surgery at Nagoya University Hospital and one glioma patient (glioblastoma, WHO grade IV), who underwent primary surgery at Tsukuba University Hospital, were included in this study.
Informed consent was obtained from each patient or the patient’s
guardian for obtaining samples and for subsequent data analysis.
2.2. Hybridoma production
GMab-m1 was produced using mouse medial iliac lymph node
methods [15]. Brieﬂy, BDF1 mice (Japan SLC Inc., Shizuoka, Japan)
were immunized by injecting 80 lg of synthetic peptides of
CGGVKPIIIGGHAYGDQYRA (IDH1-R132G), conjugated with KLH together with Freund’s complete adjuvant (FCA; Sigma–Aldrich, St.
Louis, MO) into tailbase. RcMab-1 was produced using rat medial
iliac lymph node methods [15]. WKY/Izm rats (Japan SLC Inc., Shizuoka, Japan) were immunized by injecting 170 lg of recombinant
IDH1 protein expressed in Escherichia coli TOP-10 cells into footpad. The lymphocytes were fused with mouse myeloma Sp2/0 cells
using polyethylene glycol (PEG) methods. The culture supernatants
were screened using ELISA for binding to the IDH1-R132G and
IDH1-WT peptides, conjugated with BSA.
2.3. Enzyme-linked immunosorbent assay (ELISA)
Synthetic peptides were immobilized on Nunc Maxisorp 96well immunoplates (Thermo Fisher Scientiﬁc Inc., Waltham, MA)
at 1 lg/ml for 30 min, respectively. After blocking with SuperBlock
T20 (PBS) Blocking Buffer in PBS (Thermo Fisher Scientiﬁc Inc.), theplates were incubated with culture supernatant, followed by
1:1000 diluted peroxidase-conjugated anti-mouse IgG (Dako,
Glostrup, Denmark). The enzymatic reaction was conducted with
1-Step Ultra TMB-ELISA (Thermo Fisher Scientiﬁc Inc.). The opticaldensity was measured at 655 nm with an iMark microplate reader(Bio–Rad Laboratories Inc., Philadelphia, PA). These reactions were
performed with a volume of 50 ll at 37 °C.

2.6. Western-blot analyses
Cultured cell pellets were lysed with PBS with 1% TritonX-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 10 lg of total protein were prepared for Western-blot analyses by boiling in SDS sample buffer (Nakalai Tesque, Inc.). They
were electrophoresed on 5–20% polyacrylamide gels (Wako Pure
Chemical Industries Ltd.). The separated proteins were transferred
to a PVDF membrane (Bio–Rad Laboratories Inc.). After blocking
with 4% skim milk in PBS for 15 min, the membrane was incubatedwith GMab-m1 (1 lg/ml), RMab-3 [14] (1 lg/ml), RcMab-1 (1 lg/ml), anti-MBP tag [15,16] (TMab-2; 1 lg/ml), and anti-V5 tag(1:1000 diluted; MBL, Nagoya, Japan) for 30 min; then with peroxidase-conjugated secondary antibodies (Dako, 1/1000 diluted) for
15 min, and developed with ECL-plus reagents (GE Healthcare)using SAYAKA-IMAGER (DRC Co., Ltd., Tokyo, Japan).

2.4. Plasmid preparation
Human IDH1 cDNA (GenBank Accession Nos. AF113917 or
BC012846) encoding a full length open reading frame (ORF) was
obtained by PCR using a human lung cDNA library (Cosmo BioCo., Ltd., Tokyo, Japan) as a template. The primer set for IDH1
was as follows: EcoRI-IDH1-F1, 50 -cacgaattcATGTCCAAAAAAATCA
GTGG-30 and SalI-IDH1-R1, 50 -gtggtcgacTTAAAGTTTGGCCTGAGCTA-30 . The ampliﬁed cDNA was subcloned into a pcDNA3.1/V5His-TOPO vector (Invitrogen Corp.). Substitution of the Arginine
132 (R132) to appropriate amino acid codons (Glycine or Leucine)in IDH1 cDNAs was accomplished using the QuikChange Lightning
site-directed mutagenesis kit (Agilent Technologies, Santa Clara,
CA). The full-length IDH1-WT, IDH1-R132G, and IDH1-R132L were
subcloned into an expression vector, pMAL-c2 (New England Biolabs Inc., Beverly, MA), via EcoRI and SalI restriction sites.

Fig. 1. Western-blot analyses by anti-IDH1 mAbs against MBP-fusion proteins.
Puriﬁed proteins (0.05 lg/lane) of E. coli-expressing IDH1 wild type (WT; lane 1)
and IDH1 mutants (lane 2, IDH1-R132G; lane 3, IDH1-R132L) were electrophoresed
under reducing conditions using 5–20% gels, and were Western-blotted with GMabm1, RMab-3, RcMab-1, and anti-MBP tag (TMab-2).

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Y. Kato et al. / Biochemical and Biophysical Research Communications 432 (2013) 564–567

Fig. 2. Western-blot analyses by anti-IDH1 mAbs against mutated IDH1-expressing
CHO cells. Total cell lysate from CHO cells expressing IDH1 wild type (WT; lane 1)
and IDH1 mutants (lane 2, IDH1-R132G; lane 3, IDH1-R132L) were electrophoresed
under reducing conditions using 5–20% gels, and were Western-blotted with GMabm1, RMab-3, RcMab-1, and anti-V5 tag.

2.7. Immunohistochemical (IHC) analysis
IDH1-R132G protein expression was determined immunohistochemically in parafﬁn-embedded tumor specimens. Brieﬂy, 4-lmthick histologic sections were deparafﬁnized in xylene and rehydrated. Then, they were autoclaved in citrate buffer (pH 6.0; Dako)for 20 min. Sections were incubated with 5 lg/ml of GMab-m1
over night at 4 °C. Biotin-conjugated secondary anti-mouse IgG(Dako) was incubated for 30 min at room temperature followedby the peroxidase-conjugated biotin-streptavidin complex (Vectastain ABC Kit; Vector Laboratories Inc., Burlingame, CA) for 30 min
at room temperature. Color was developed using 3, 3-diaminobenzidine tetrahydrochloride (DAB; Dako) for 10 min, andcounterstained with hematoxylin.
3. Results and discussion
3.1. Production of IDH1-R132G-speciﬁc antibodies
Speciﬁc antibodies against R132G have not been reported,
although we previously established two anti-mutated IDH1 mAbs:
HMab-1 [14] against IDH1-R132H and SMab-1 [13] against IDH1R132S. In this study, mice were immunized with synthetic peptides of IDH1-R132G, and the wells of hybridomas, which produced
IDH1-R132G-reactive/IDH1-wild type (WT)-nonreactive antibodies, were screened in ELISA. After limiting dilution, clone GMabm1 (mouse IgG2a, kappa) against IDH1-R132G was established.
GMab-m1 was shown to be an IDH1-R132G-speciﬁc mAb, notreacting with IDH1-WT, using ELISA against both synthetic peptides and recombinant proteins (data not shown).
3.2. Speciﬁcity of GMab-m1 against IDH1 mutants
To determine the speciﬁcity of GMab-m1, the reactivity against
IDH1-WT and IDH1 mutants (MBP-IDH1-R132G and MBP-IDH1R132L) were investigated using Western-blot analyses. Fig. 1
shows that GMab-m1 recognized only IDH1-R132G protein, notthe other proteins (IDH1-WT and IDH1-R132L), indicating that
GMab-m1 is a speciﬁc antibody against IDH1-R132G protein. In

Fig. 3. Immunohistochemical analyses by GMab-m1 against glioma tissues. Glioma
tissues possessing IDH1-R132G, (A and B) or IDH1-WT, (C) were stained with
GMab-m1. Magniﬁcation: 200.

contrast, RMab-3 [14] and RcMab-1 recognized IDH1-WT, IDH1R132G, and IDH1-R132L proteins. These results indicate that
GMab-m1 is useful for detecting IDH1-R132G. Another GST-tag
was also used in the expression of IDH1 mutants: GST-IDH1-WT,
GST-IDH1-R132G and GST-IDH1-R132L. As a result, GST-tagged
IDH1-WT and IDH1 mutants were also detected by anti-IDH1
mAbs totally in the same way as MBP-tagged proteins (data notshown), suggesting that the reactivity of those antibodies is observed independently of those tag proteins.
Next, we performed Western-blot analyses against mutated
IDH1-expressing CHO cells. As shown in Fig. 2, GMab-m1 recognized only IDH1-R132G protein expressed in CHO cells, not theother proteins (IDH1-WT and IDH1-R132L), indicating that
GMab-m1 is useful for detecting IDH1-R132G expressed in
mammalian cells. In contrast, RcMab-1 equally recognized

Y. Kato et al. / Biochemical and Biophysical Research Communications 432 (2013) 564–567

IDH1-WT, IDH1-R132G, and IDH1-R132L, whereas RMab-3 detected IDH1-WT strongly. RcMab-1 and RMab-3 also detects
endogenous hamster IDH1.
3.3. Immunohistochemical (IHC) analysis by GMab-m1 against IDH1R132G-bearing gliomas
We next performed immunohistochemistry of GMab-m1
against IDH1-R132G-positive gliomas. Typical results are shownin Fig. 3. GMab-m1 stained almost all tumor cells of IDH1R132G-positive gliomas (Fig. 3A), although no staining was observed in IDH1-WT gliomas (Fig. 3C). Further, GMab-m1 did notstain endothelial cells in gliomas (Fig. 3B). These results indicatethat GMab-m1 is useful in immunohistochemical analyses for
detection of IDH1-R132G mutation. In near future, combinationof GMab-m1 with previously established anti-mutated IDH1 mAbs
(HMab-1 and SMab-1) should increase the diagnostic rate using
immunohistochemistry against glioma patients.
In conclusion, the newly established anti-IDH1-R132G-speciﬁc
mAb, GMab-m1, is expected to be extremely useful for diagnosis
and biological evaluation of IDH1-R132G-bearing gliomas in combination with previously established anti-mutated IDH1-speciﬁc
monoclonal antibodies (HMab-1 and SMab-1). Additional IDH1
mutation-speciﬁc antibodies such as anti-IDH1-R132C must be
developed for pathological diagnosis of gliomas, cartilaginous tumors, and acute myeloid leukemias.
Acknowledgments
We thank Fumiharu Ohka (Nagoya University) and Shingo Takano (University of Tsukuba) for providing us glioma specimen. We
also thank Yuta Tsujimoto, Shunpei Morita, Ryo Yanagiya, KanaNasu, Hiroko Sasaki, Sachiko Suzuki, and Junko Aita (Yamagata
University) for their excellent technical assistance. This work was
supported in part by Platform for Drug Discovery, Informatics,
and Structural Life Science (M.K.K., Y.K.), by Regional Innovation
Strategy Support Program (Y.K.), and by KAKENHI (23790185,
23701043): a Grant-in-Aid for Young Scientists (B) (M.K.K., Y.K.)
from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by Intelligent Cosmos Academic Foundation(Y.K.).
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