Biochemical and Biophysical Research Communications 390 (2009) 547–551

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

A monoclonal antibody IMab-1 speciﬁcally recognizes IDH1R132H,
the most common glioma-derived mutation
Yukinari Kato a,b,*, Genglin Jin a, Chien-Tsun Kuan a, Roger E. McLendon a, Hai Yan a, Darell D. Bigner aab

Department of Pathology, Duke University Medical Center, DUMC-3156, Durham, NC 27710, USA
The Oncology Research Center, Research Institute for Advanced Molecular Epidemiology, Yamagata University, 2-2-2 Iida-nishi, Yamagata 990-9585, Japana r t i c l e

i n f oArticle history:
Received 30 September 2009
Available online 7 October 2009
Keywords:
IDH1
Mutation
R132H
Monoclonal antibody
Astrocytoma
Glioblastomaa b s t r a c t
IDH1 (isocitrate dehydrogenase 1) mutations have been identiﬁed as early and frequent genetic alterationsin astrocytomas, oligodendrogliomas, and oligoastrocytomas as well as secondary glioblastomas. In
contrast, primary glioblastomas very rarely contain IDH1 mutations, although primary and secondary glioblastomas are histologically indistinguishable. The IDH1 mutations are remarkably speciﬁc to a single
codon in the conserved and functionally important Arg132 in IDH1. In gliomas, the most frequent IDH1
mutations (>90%) were G395A (R132H). In this study, we immunized mice with R132H-containing IDH1
(IDH1R132H) peptide. After cell fusion using Sendai virus envelope, the monoclonal antibodies (mAbs), which
speciﬁcally reacted with IDH1R132H, were screened in ELISA. One of the mAbs, IMab-1 reacted with the
IDH1R132H peptide, but not with wild type IDH1 (IDH1wt) peptide in ELISA. In Western-blot analysis,
IMab-1 reacted with only the IDH1R132H protein, not IDH1wt protein or the other IDH1 mutants, indicatingthat IMab-1 is IDH1R132H-speciﬁc. Furthermore, IMab-1 speciﬁcally stained the IDH1R132H-expressing cells
in astrocytomas in immunohistochemistry, whereas it did not react with IDH1R132H-negative primary
glioblastoma sections. In conclusion, we established an anti-IDH1R132H-speciﬁc monoclonal antibody
IMab-1, which should be signiﬁcantly useful for diagnosis and biological evaluation of mutation-bearing
gliomas.
Ó 2009 Elsevier Inc. All rights reserved.

Introduction
Gliomas are the most common primary brain tumors, and aregrouped into four grades (grade I–IV) according to the World
Health Organization (WHO) criteria. Among them, glioblastomas(GBMs) are the most frequent and malignant type of gliomas [1].
Despite advances in surgical techniques, radiation therapy andadjuvant chemotherapy, their prognoses remain poor: the mediansurvival time for patients with GBMs is only one year [2]. GBMs
may occur de novo (primary GBM) or may result from progressionof low-grade astrocytomas (secondary GBM) [3,4].
An unbiased, genome-wide analysis of the somatic mutations
occurring in GBMs revealed recurrent mutations in arginine(amino acid position 132: R132), which is the active site of
IDH1, in 12% of tumors analyzed [5]. In the subsequent genetic
studies, IDH1 has been found to be mutated in 50–88% of secondary GBMs, whereas only 3–12% of primary GBMs possess theseAbbreviations: IDH1, isocitrate dehydrogenase 1; WHO, World Health Organization; CHO, Chinese hamster ovary.
* Corresponding author. Address: Department of Pathology, Duke University
Medical Center, DUMC-3156, Durham, NC 27710, USA. Fax: +1 919 684 6791.
E-mail address: yukinari-k@bea.hi-ho.ne.jp (Y. Kato).
0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2009.10.001

alterations [6–13]. IDH1 seems to function as a tumor suppressor;
therefore, mutated IDH1 contributes to tumorigenesis in partthrough induction of the HIF-1 pathway [14]. Analyses of progressive glioma samples, including malignant diffuse astrocytomas,
anaplastic astrocytomas, well-differentiated oligodendrogliomas,
anaplastic oligodendrogliomas, and the mixed oligoastrocytomas,
and anaplastic oligoastrocytomas revealed that mutations in
IDH1-R132 (IDH1R132H) are in fact common (>70%) in these tumors, and also occur less frequently in primary glioblastomas
and other cancers [6–13]. Furthermore, the IDH1 mutation was
only rarely detected in any WHO grade I pilocytic astrocytomas[6,7,9,15], a tumor type with infrequent malignant progression,indicating that these tumors arise through a different mechanism.
A monoclonal antibody, which can speciﬁcally recognize the
IDH1R132 is urgently needed for both research and clinical purposes. Furthermore, an immunohistochemical assay would be
advantageous in the evaluation of the diagnosis and prognosis
of a glioma biopsy as well as for biological studies such as evaluating the extent of invasion in mutation-bearing gliomas. In thisstudy, we report a monoclonal antibody, IMab-1, which speciﬁcally detects IDH1R132H in ELISA, Western-blot analysis, andimmunohistochemistry.

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Y. Kato et al. / Biochemical and Biophysical Research Communications 390 (2009) 547–551

Materials and methods
Animals, cell lines, and tissues. Female BALB/c mice were obtained from The Jackson laboratory (Bar Harbor, Maine). Chinese
hamster ovary (CHO) and P3U1 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA). CHO and P3U1
were cultured at 37 °C in a humidiﬁed atmosphere of 5% CO2 and
95% air in RPMI 1640 medium including 2 mM L-glutamine (Invitrogen Corp., Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma, St. Louis, MO) and 1% of
penicillin–streptomycin solution (Invitrogen Corp.). HOG cells
were cultured as described previously [7]. Human tissues slides
were obtained from the Tissue Bank at the Preston Robert TischBrain Tumor Center at Duke University as described previously[7]. Four anaplastic astrocytoma (WHO Grade III) tissues slides
and one diffuse astrocytoma (WHO Grade II) tissue slides, which
possess IDH1-R132H mutation (IDH1R132H), and two glioblastoma(WHO grade IV) tissues, which do not possess IDH1R132H, were previously analyzed [7].
Hybridoma production. BALB/c mice were immunized by neck s.c.
injections of 100 lg of synthetic peptide KPIIIGHHAYGDQYRC(IDH1R132H peptide), corresponding to amino acids 126–140 of
the human IDH1R132H, plus C-terminus cystein conjugated with
KLH with Imject Freund’s Complete Adjuvant (Thermo Scientiﬁc,Inc., Rockford, IL). One week later, secondary i.p. immunization of
100 lg of IDH1R132H peptide was performed. After additional
immunization of 100 lg of IDH1R132H peptide, the booster injection
was given i.p. 2 days before spleen cells were harvested. The spleencells were fused with mouse myeloma P3U1 cells using Sendai virus(Hemagglutinating Virus of Japan: HVJ) envelope: GenomONE-CFEX(Cosmo Bio USA, Inc., Carlsbad, CA) according to the manufacturers’
instructions. The hybridomas were grown in RPMI medium including hypoxanthine, aminopterin and thymidine selection medium
supplement (Sigma), 2 mM L-glutamine (Invitrogen Corp.), 10%
heat-inactivated FBS (Sigma), 5% BriClone (QED Bioscience Inc.,San Diego, CA), and 1% of penicillin-streptomycin, solution (Invitrogen Corp.). The culture supernatants were screened by ELISA for thebinding to the IDH1R132H peptide and the IDH1-wild type (IDH1wt)
as shown in ELISA section. Single cell cloning was performed using
ClonaCell-HY Hybridoma Selection Medium (Medium D; StemCell
Technologies Inc., Vancouver, Canada).
Enzyme-linked immunosorbent assay (ELISA). Synthetic peptides
corresponding to amino acids 126–140 of the human IDH1wt:(KPIIIGRHAYGDQYRC) and IDH1R132H (KPIIIGHHAYGDQYRC) plus
C-terminus cystein conjugated with BSA were immobilized on
96-well plates at 1 lg/ml for 30 min, respectively. After blocking
with 1% BSA in PBS, the plates were incubated with IMab-1 at several concentrations, followed by 1:1000 diluted peroxidase-conjugated anti-mouse IgG (GE Healthcare UK Ltd., Buckinghamshire,
England). The enzymatic reaction was conducted with a substratesolution containing TMB (Thermo Scientiﬁc, Inc.). After the reaction was stopped with 2 M H2SO4, the optical density was measured at 450 nm with an auto-plate reader (Bio-Rad Laboratories,
Inc., Philadelphia, PA). These reactions were performed with a volume of 50 ll at room temperature.
Establishment of stable transfectants of IDH1wt and IDH1R132H.
CHO cells were transfected with vectors (pCMV6, Invitrogen Corp.)
containing the coding sequences of IDH1wt and IDH1R132H [7] using
LipofectAMINE 2000 reagent (Invitrogen Corp.) according to the
manufacturers’ instructions. Stable CHO transfectants were selected by cultivating the transfectants in medium containing
1 mg/ml of geneticin (Invitrogen Corp.), and the expression levelsof IDH1wt and IDH1R132H were conﬁrmed by Western-blot analysis.
Western-blot analysis. Stable CHO transfectants of IDH1wt and
IDH1R132H were lysed with lysis buffer (1% Triton X-100 in PBS con-Fig. 1. Production of a speciﬁc monoclonal antibody against IDH1R132H. (A)Synthetic peptides corresponding to amino acids 126–140 of the human IDH1wt
and IDH1R132H plus C-terminus cystein conjugated with BSA were immobilized on
96-well plates. After blocking, the plates were incubated with IMab-1 at several
concentrations, followed by peroxidase-conjugated anti-mouse IgG. The enzymatic
reaction was conducted with a substrate solution containing TMB. After thereaction was stopped with 2 M H2SO4, the optical density was measured at 450 nm.
(B) Cell lysates from CHO-stable transfectants expressing IDH1wt or IDH1R132H were
electrophoresed under reducing conditions using 4–10% gels, and Western-blottedwith anti-FLAG, anti-IDH1, IMab-1, and anti-b-actin.

taining 50 mg/ml aprotinin). HOG cells were transfected with
pCMV6 vectors containing the coding sequences of IDH1wt and ﬁve
IDH1 mutants, and lysed as described previously [7]. Samples ofthe supernatant fraction were collected after centrifuging at
15,000g for 5 min. Ten micrograms of the proteins were electrophoresed under reducing conditions on 4–10% NuPAGE gel (Invitrogen). The separated proteins were transferred to a PVDF
membrane (Bio-Rad Laboratories, Inc.). After blocking with 5%
skim milk in PBS with 0.05% Tween 20, the membrane was incubated with mouse anti-FLAG (M2; 1 lg/ml; Sigma), goat antiIDH1 (1/200 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz,
CA), IMab-1 (1 lg/ml), mouse anti-Myc (1/2000; Origene Technologies, Inc., Rockville, MD), mouse anti-b-actin antibody (1/5000
dilution; Sigma), or rabbit anti-GAPDH (1/4000 dilution; Santa
Cruz Biotechnology), and subsequently with peroxidase-conjugated anti-goat (1/40,000 dilution; Sigma), anti-rabbit (1/5000
dilution; GE Healthcare UK Ltd.) or anti-mouse antibodies(1/25,000 dilution; GE Healthcare UK Ltd.). It was then developedfor 5 s to 1 min with ECL (GE Healthcare UK Ltd.), ECL-plus

Y. Kato et al. / Biochemical and Biophysical Research Communications 390 (2009) 547–551

reagents (GE Healthcare UK Ltd.), or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientiﬁc) using Amersham
Hyperﬁlm ECL (GE Healthcare UK Ltd.).
Immunohistochemical analysis. All procedures were performed
using Bond Polymer Reﬁne Detection of BondTM Automated Immunohistochemistry system (Leica Microsystems, Inc., Bannockburn,IL). Brieﬂy, formalin ﬁxed, parafﬁn embedded 5 lm thick sections
were deparafﬁnized, rehydrated, and heated at 100 °C using citratebuffer (pH 6.0) for 5 min, and incubated ﬁrst with IMab-1 (5 lg/ml)
for 15 min, then with post-primary blocker for 8 min, and ﬁnallywith polymer for 8 min. Color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB) for 10 min, and counterstained by hematoxylin.

549

Fig. 2. IMab-1 reacts with only IDH1R132H. Cell lysates from HOG transienttransfectants expressing IDH1wt and ﬁve IDH1 mutants were electrophoresed
under reducing conditions using 4–10% gels, and Western-blotted with anti-Myc,
IMab-1, and anti-GAPDH.

Results
Production of an anti-IDH1R132H-speciﬁc antibody
We immunized mice with synthetic peptides of IDH1-R132H
mutant (IDH1R132H). After cell fusion using Sendai virus envelope,
the wells of hybridomas, which were producing anti-IDH1R132H-

speciﬁc antibodies, were selected in ELISA. After limiting dilution,
one of the clones, IMab-1 (IgG1 subclass) was established. As
shown in Fig. 1A, IMab-1 reacted with IDH1R132H peptide in adose-dependent manner, but not with IDH1-wild type (IDH1wt)
in ELISA, indicating that IMab-1 speciﬁcally recognized IDH1R132H.

Fig. 3. Immunohistochemical analysis by IMab-1 against glioma tissues with IDH1R132H. Anaplastic astrocytoma tissues which have IDH1R132H (A, B, and E), diffuse
astrocytoma tissues which have IDH1R132H (C, D), and primary glioblastoma tissues which have no IDH1R132H (F) were stained by IMab-1 (A, C, E, and F) or isotype control(B, D). Magniﬁcation: 200.

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Y. Kato et al. / Biochemical and Biophysical Research Communications 390 (2009) 547–551

Furthermore, IDH1wt and IDH1R132H proteins were stably expressed in Chinese hamster ovary (CHO) cells, and conﬁrmed byWestern-blot analysis (Fig. 1B). Both anti-IDH1wt and anti-FLAG
antibodies detected IDH1wt and IDH1R132H proteins at the same
expression level. Endogenous IDH1wt was also detected slightly
by anti-IDH1wt antibody in both stable transfectants. Importantly,
IMab-1 recognized only IDH1R132H, not IDH1wt. These results indicate that IMab-1 is also useful in detecting not only IDH1R132H peptide, but also the IDH1R132H protein.
Speciﬁcity of IMab-1 against IDH1 mutants
All IDH1 mutants reported until now were transiently transfected into HOG cells (Fig. 2). IMab-1 recognized only IDH1R132H, notthe other mutants: IDH1R132C, IDH1R132G, IDH1R132L, and IDH1R132S,
indicating that IMab-1 is a speciﬁc antibody against IDH1R132H.
Immunohistochemical analysis by IMab-1 against glioma tissues with
IDH1R132H
IMab-1 was conﬁrmed to be an anti-IDH1R132H-speciﬁc antibody
in ELISA and Western-blot analysis; therefore, we ﬁrst performed
immunohistochemistry using CHO-stable transfectants of IDH1wt
or IDH1R132H, which were embedded in parafﬁn. Using those parafﬁn sections, we optimized conditions of immunohistochemistry.
IMab-1 reacted with only IDH1R132H-transfectant parafﬁn sectionsin the conditions as described in Materials and methods (data notshown). We next performed immunohistochemistry of IMab-1
against several IDH1R132H-positive gliomas and IDH1R132H-negative
gliomas, which were previously characterized [7]. Typical results
were shown in Fig. 3. IMab-1 stained almost all tumor cells of
IDH1R132H-positive gliomas (Fig. 3A, C, and E), whereas no staining
was observed in IDH1R132H-negative gliomas (Fig. 3F). IMab-1 did
not stain endothelial cells or red blood cells in IDH1R132H-positive
gliomas (Fig. 3E). The concentration-matched isotype control ofmouse IgG1 did not stain any tumor cells (Fig. 3B and D), althoughat higher concentrations, non-speciﬁc binding of gemistocytes is
encountered with this isotype control antibody (data not shown).
These results indicate that IMab-1 is useful in immunohistochemistry for detection of IDH1R132H mutation.

Discussion
The IDH1 gene at 2q33 encodes isocitrate dehydrogenase 1(IDH1), which catalyzes the oxidative carboxylation of isocitrate
to a-ketoglutarate, which leads to the production of NADPH in
the citric acid cycle [14]. IDH1 mutations occur frequently in some
types of malignant gliomas, and less frequently in other gliomas[7]. To date, all mutations have been identiﬁed at the critical isocitrate binding site R132 and the vast majority of changes are heterozygous [6–13]. In gliomas, mutations in the IDH1 homolog
IDH2 have been identiﬁed at the aligned R172 [7]. These discoveries mark unique ﬁndings in the ﬁeld of cancer genetics. In thisstudy, we produced an anti-IDH1-R132H (IDH1R132H)-speciﬁc antibody, IMab-1. IMab-1 did not react with wild type of IDH1
(IDH1wt) or any other IDH1 mutants in ELISA and Western-blot
analysis (Figs. 1 and 2). Importantly, using IMab-1, we have developed an immunohistochemical method which allows us to view
IDH1R132H-positive cells in the clinical samples. IMab-1 displays
speciﬁc cytosolic staining pattern in the tumor cells, whereasshows no staining in normal cells such as endothelial cells or blood
cells (Fig. 3). IDH1 mutations were reported to be very early events,
before TP53 mutations or loss of 1p/19q occur [10], which may leadto diffused staining by IMab-1 in almost every single cell of
IDH1R132H-positive gliomas.

IDH1 is mutated in the majority of secondary GBMs, whereas
less than 10% of primary GBMs possess these alterations [7,10].
IMab-1 can serve as a molecular tool for distinguishing subtypesof GBMs. Furthermore, IMab-1 could help distinguish pilocytic
astrocytomas (WHO grade I), which rarely express IDH1R132H fromdiffuse astrocytomas (WHO grade II), since these lesions can sometimes be difﬁcult to categorize solely on the basis of histopathological criteria [15]. Most recently, IDH1 mutation have been foundin 15 of 187 acute myeloid leukemia samples (AML) [16]. The othertumors, in which IDH1 mutations are reported, are one of colorectal cancer [17], two prostate cancers [18], one B-acute lymphoblastic leukemia (B-ALL) [18], and three adult supratentorial primitive
neuroectodermal tumors (sPNET) [6]. IMab-1 might be also usefulfor diagnosis of these kinds of IDH1R132H-positive tumors. The
R132C mutations (IDH1R132C) were observed at high frequency in
AML (8/16 IDH1 mutations; 50%), whereas it was observed in only
4% in glioma specimens [16]. In contrast, IDH1R132H was observedin only 44% of IDH1 mutations in AML, whereas it was observedin 88% in glioma specimens [7]. These results suggest that an
anti-IDH1R132C-speciﬁc antibody may be useful for diagnosis of
AML as well as glioma subtypes.
Therefore, the established anti-IDH1R132H-speciﬁc monoclonal
antibody IMab-1 should be signiﬁcantly useful for diagnosis andbiological evaluation of mutation-bearing gliomas.

Acknowledgments
We thank Mr. Ling Wang, Mr. Charles Pegram, and Mr. Scott E.
Szafranski for technical assistance, Dr. Joanne Ayriss, Dr. Jinli
Chang, Dr. Mika Kato Kaneko, and Dr. Chifumi Kitanaka for theirgreat help. This study was supported by NIH Grants: 5-P5010876, 5-P50-NS-20023, CA11898, and 5-R01-CA118822. Y.K.
was supported by the Global COE Program for Medical Sciences, Japan Society for the Promotion of Science.

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