MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 32, Number 3, 2013ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2013.0006

Short Communications

Novel Monoclonal Antibodies GMab-r1 and LMab-1
Specifically Recognize IDH1-R132G
and IDH1-R132L Mutations
Mika Kato Kaneko,1,2* Yuta Tsujimoto,2* Yasukazu Hozumi,3 Kaoru Goto,3 and Yukinari Kato1,2

Isocitrate dehydrogenase 1 (IDH1) catalyzes the oxidative carboxylation of isocitrate to a-ketoglutarate in cytosol. IDH1 mutations, which are specific to a single codon in the conserved and functionally important Arginine
132 (R132), result in the ability of the enzyme to catalyze the reduced NADP-dependent reduction of aketoglutarate to onco-metabolite R(-)-2-hydroxyglutarate (2-HG). IDH1 mutations, which are early and frequent
genetic alterations that occur in gliomas, cartilaginous tumors, and leukemias. We previously established two
monoclonal antibodies (MAbs) that are specific for IDH1 mutations: clone HMab-1 against IDH1-R132H andclone SMab-1 against IDH1-R132S. However, specific MAbs against IDH1-R132G or IDH1-R132L have not beenreported. To establish IDH1-R132G-specific or IDH1-R132L-specific MAbs, we immunized rats with eachmutation-containing IDH1 peptides, and IDH1-R132G-specific or IDH1-R132L-specific MAbs were screened in
ELISA. Established MAb GMab-r1 reacted with the IDH1-R132G peptide, but not with IDH1-wild type (WT) in
ELISA. In contrast, LMab-1 reacted with the IDH1-R132L peptide, but not with IDH1-WT. Western blot analysis
also showed that GMab-r1 and LMab-1 reacted with the IDH1-R132G and IDH1-R132L recombinant proteins,respectively, but not with IDH1-WT or other IDH1 mutants, indicating that GMab-r1 and LMab-1 are IDH1mutation-specific. Furthermore, GMab-r1 and LMab-1 specifically stained the IDH1-R132G- and IDH1-R132Lexpressing cells in immunocytochemistry, respectively. This is the first report to establish anti-IDH1-R132G-specificor IDH1-R132L-specific MAbs, which could be useful in the diagnosis of mutation-bearing tumors.

IntroductionT

he IDH1 gene at 2q33 encodes isocitrate dehydrogenase
1 (IDH1), which catalyzes the oxidative carboxylationof isocitrate to a-ketoglutarate. IDH1 mutations were found toresult in the ability of the enzyme to catalyze the reduced NADPdependent reduction of a-ketoglutarate to onco-metabolite R(-)2-hydroxyglutarate (2-HG).(1) 2-HG is reported to accumulate in
the inherited metabolic disorder 2-hydroxyglutaric aciduria,
which is caused by deficiency of 2-hydroxyglutarate dehydrogenase, because 2-hydroxyglutarate dehydrogenase converts
2-HG to a-ketoglutarate.(2) Patients with 2-hydroxyglutarate
dehydrogenase deficiencies are known to accumulate 2-HG in
the brain, develop leukoencephalopathy, and possess an increased risk of brain tumors. Moreover, elevated 2-HG levels inbrain result in increased ROS levels, potentially contributing toan increased risk of cancer.(3)IDH1 mutations occur in some malignant gliomas,(4) cartilaginous tumors,(5,6) and acute myeloid leukemias.(7) In as-

trocytomas, oligodendrogliomas, oligoastrocytomas, andsecondary glioblastomas, IDH1 mutations have been identified as early and frequent genetic alterations (50–93%), andmight be the initiating event in these glioma subtypes.(4,8–10)
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. Hartmann and colleagues reported that IDH1 mutations include R132H (664/716: 92.7%), R132C (29/716: 4.2%),
R132S (11/716: 1.5%), R132G (10/716: 1.4%), and R132L (2/716: 0.2%).(11) Another R132V mutation was reported in 1212
IDH1 mutations (1/1212: 0.1%) by von Deimling and colleagues.(12) To date, several monoclonal antibodies (MAbs)
against IDH1 mutations have been reported.(13–17) Of
those antibodies, we established IDH1-R132H-specific MAb
HMab-1(16) and IDH1-R132S-specific MAb SMab-1.(15) HMab1 reacted with the IDH1-R132H peptide, but not with the1

Regional Innovation Strategy Support Program, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.
Molecular Tumor Marker Research Team, Global COE Program, Yamagata University Faculty of Medicine, Yamagata, Japan.
3
Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata, Japan.
*These authors contributed equally to this work.
2

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225

wild-type IDH1 (IDH-WT) peptide in ELISA. In Western blot
analysis, HMab-1 reacted only with the IDH1-R132H protein,
not with the IDH1-WT protein or other IDH1 mutants. SMab1 reacted with IDH1-R132S specifically in the same way.(15) In
our recent study, 164 cases of glioma were evaluated immunohistochemically for IDH1 mutations (R132H and R132S)
using anti-IDH1 MAbs HMab-1 and SMab-1.(13) IDH1 mutation was detected, respectively, in 9.7%, 63.6%, 51.7%, and77.8% of primary grade IV, secondary grade IV, grade III, andgrade II gliomas. IDH1 mutation as evaluated using immunohistochemistry might be of greater prognostic significancethan histological grading alone in grade III and grade IV gliomas.
Although HMab-1 and SMab-1 are able to find more than
90% of IDH1 mutations of gliomas in immunohistochemistry,other IDH1 mutations have been missed unfortunately inimmunohistochemistry. Therefore, novel antibodies that recognize other IDH1 mutations should be developed to cover allof IDH1 mutations in immunohistochemistry. Here, we
newly report anti-IDH1-mutation-specific monoclonal antibodies (GMab-r1 and LMab-1) that are expected to be extremely useful for diagnosis of mutation-bearing gliomas.

Scientific). The optical density was measured at 655 nm with
an iMark microplate reader (Bio-Rad Laboratories, Philadelphia, PA). These reactions were performed with a volume of
50 mL at 37!C.
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., Tokyo, Japan) as a template. The primer set
for IDH1 was as follows: EcoRI-IDH1-F1, 5¢-cacgaattc
ATGTCCAAAAAAATCAGTGG-3¢; and SalI-IDH1-R1, 5¢gtggtcgacTTAAAGTTTGGCCTGAGCTA-3¢. The amplified
cDNA was subcloned into a pcDNA3.1/V5-His-TOPO vector(Life Technologies Corp.). Substitution of the Arginine 132(R132) to appropriate amino acid codons (Glycine or Leucine)in IDH1 cDNAs was accomplished using the QuikChangeLightning site-directed mutagenesis kit (Stratagene, La Jolla,
CA). The full-length IDH1-WT, IDH1-R132G, and IDH1R132L were subcloned into an expression vector, pMAL-c2(New England Biolabs, Beverly, MA) via EcoRI and SalI restriction sites.

Materials and Methods
Cell lines
Chinese hamster ovary (CHO) cells and Sp2/0 were obtained from the American Type Culture Collection (ATCC,
Manassas, VA). These cell lines were cultured at 37!C in ahumidified atmosphere of 5% CO2 and 95% air in RPMI 1640
medium (Wako Pure Chemical Industries, Osaka, Japan), respectively, including 2 mM L-glutamine (Wako Pure Chemical Industries) supplemented with 10% heat-inactivated fetal
bovine serum (FBS; Life Technologies Corp., Carlsbad, CA).
Hybridoma production
Three rat monoclonal antibodies (GMab-r1, LMab-1, and
RcMab-1) were produced using rat medial iliac lymph
node methods.(18–21) Briefly, WKY/Izm rats were immunized
by injecting 170 mg of synthetic peptides of CGGVKPIIIGG
HAYGDQYRA (IDH1-R132G) for GMab-r1 or CGGVKPIIIGLHAYGDQYRA (IDH1-R132L) for LMab-1, conjugated with
KLH together with Freund’s complete adjuvant (FCA; SigmaAldrich, St. Louis, MO) or recombinant IDH1 protein for
RcMab-1(20) into footpad. The lymphocytes were fused withmouse myeloma Sp2/0 cells using polyethylene glycol (PEG)
methods. The culture supernatants were screened using ELISA for binding to the IDH1 mutations (IDH1-R132G or IDH1R132L) and IDH1-WT peptides conjugated with BSA for
GMab-r1 and LMab-1 or recombinant IDH1 protein for
RcMab-1.
Enzyme-linked immunosorbent assay
Synthetic peptides were immobilized, respectively, on
Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific, Waltham, MA) at 1 mg/mL for 30 min, respectively.
After blocking with 1% BSA (Sigma-Aldrich) in PBS, theplates were incubated with culture supernatant, followed by
1:1000 diluted peroxidase-conjugated anti-rat IgG (Dako,
Glostrup, Denmark). The enzymatic reaction was conductedwith a substrate solution containing TMB (Thermo Fisher

Protein expression using bacteria cells
and mammalian cells
Competent Escherichia coli TOP-10 cells (Life Technologies
Corp.) were transformed with the plasmid, pMAL-IDH1-WT,
pMAL-IDH1-R132G, and pMAL-IDH1-R132L. They were
cultured overnight at 37!C in LB medium (Life Technologies)
containing 100 mg/mL ampicillin (Sigma-Aldrich). Cell pellets
were resuspended in phosphate-buffered saline (PBS) with
1% Triton X-100 with 50 mg/mL aprotinin (Sigma-Aldrich).
After sonication, the crude extracts were collected by centrifugation (9000 g, 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 eluted bycolumn buffer with 10 mM maltose. CHO cells were transfected with appropriate amounts of plasmids, pcDNA3.1/
IDH1-WT, pcDNA3.1/IDH1-R132G, pcDNA3.1/IDH1R132L using Lipofectamine LTX (Life Technologies Corp.)
according to the manufacturer’s instructions. Stable transfectants were selected by cultivating the transfectants in medium
containing 1 mg/mL of G418 (Wako Pure Chemical Industries). The expression level of IDH1 was confirmed using
Western blot analysis.
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 10 mg of total protein were prepared forWestern blot analysis by boiling in SDS sample buffer (Nakalai Tesque, Kyoto, Japan). They were electrophoresed on 5–
20% polyacrylamide gels (Wako Pure Chemical Industries).
The separated proteins were transferred to a PVDF membrane(EMD Millipore Corp., Billerica, MA). After blocking with 4%
skim milk in PBS for 15 min, the membrane was incubatedwith GMab-r1 (1 mg/mL), LMab-1 (1 mg/mL), RcMab-1 (1 mg/mL), anti-V5 tag (1:1000 dilution; MBL, Nagoya, Japan), anti-

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KANEKO ET AL.

MBP (TMab-2; 1 mg/mL),(19) or anti-GST tag (1:1000 dilution;
Wako Pure Chemical Industries) for 15 min, then with peroxidase-conjugated secondary antibodies (1:1000 dilution;Dako) for 15 min, and developed with Pierce Western Blotting
Substrate Plus (Thermo Fisher Scientific, Inc.) using SAYACAIMAGER (DRC Co., Tokyo, Japan).
Immunocytochemical analyses
Cultured cells were fixed with 4% paraformaldehyde in
0.1 M phosphate buffer (pH 7.4) for 20 min at room temperature, and 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 non-specific binding sites, andwere incubated with 5 mg/mL of GMab-r1, LMab-1, or
RcMab-1 containing 0.1% Triton X-100 overnight at 4!C in amoist chamber. They were incubated with goat rat IgG-Alexa
488 (1:400 dilution; Molecular Probes, Eugene, OR) in PBS
containing 0.1% Triton X-100 for 1 h at room 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, Jena,
Germany). Then fluorescent images were processed using
image-processing software (Adobe Photoshop, Adobe Systems, San Jose, CA).
Results and Discussion
Production of IDH1-R132G-specificand IDH1-R132L-specific antibodies
We previously established two anti-mutated IDH1 MAbs:
HMab-1(13) against IDH1-R132H and SMab-1(15) against
IDH1-R132S. However, specific antibodies against R132G or
R132L have not been reported. In this study, we immunized
rats with synthetic peptides of IDH1-R132G or IDH1-R132L.
After cell fusion, the wells of hybridomas, which produced
IDH1-R132G-reactive/IDH1-wild type (WT)-nonreactiveor IDH1-R132L-reactive/IDH1-WT-nonreactive antibodies,were screened in ELISA. After limiting dilution, clone
GMab-r1 (rat IgG2a subclass) against IDH1-R132G and clone
LMab-1 (rat IgG2a subclass) against IDH1-R132L were established. One anti-IDH1-WT MAb, clone RcMab-1 (rat
IgG2a), was also established. GMab-r1 and LMab-1 wereshown to be IDH1-mutation-specific MAbs, not reactingwith IDH1-WT, using ELISA against both synthetic peptides
and recombinant proteins (data not shown). In contrast,
RcMab-1 was shown to be reactive with both IDH1 mutants
and IDH1-WT using ELISA against both recombinant proteins (data not shown).
Specificity of anti-mutated IDH1 MAbs against
IDH1 mutants
To determine the specificity of GMab-r1, LMab-1, and
RcMab-1 MAbs, the reactivity against IDH1-WT and IDH1
mutants (MBP-IDH1-R132G and MBP-IDH1-R132L) were
investigated using Western blot analyses. Figure 1 shows that
GMab-r1 recognized only IDH1-R132G protein, not the otherproteins (IDH1-WT and IDH1-R132L), indicating that GMabr1 is specific antibodies against IDH1-R132G protein. In contrast, LMab-1 reacted with only IDH1-R132L protein, not theother proteins (IDH1-WT and IDH1-R132G), indicating that
LMab-1 is a specific antibody against IDH1-R132L protein.

FIG. 1. Western blot analyses by anti-IDH1 MAbs against
MBP-fusion proteins. Purified proteins (0.05 mg/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 GMab-r1, LMab-1, RcMab-1,
and anti-MBP tag (TMab-2).

RcMab-1 recognized IDH1-WT, IDH1-R132G, and IDH1R132L proteins. These results indicate that GMab-r1 andLMab-1 are useful in detecting IDH1-R132G or IDH1-R132L,
respectively. To exclude the possibility that the specificities ofthese MAbs were caused by MBP-tag, which was used for the
expression of IDH1 mutants in E. coli; another GST-tag was
also used in the expression of IDH1 mutants GST-IDH1-WT,
GST-IDH1-R132G, and GST-IDH1-R132L. Results show that
GST-tagged IDH1-WT and IDH1 mutants were also detected
totally in the same way as MBP-tagged proteins (data notshown), suggesting that the reactivity of those antibodies isobserved independently of those tag proteins.
We next performed Western blot analyses against mutated
IDH1-expressing CHO cells. As shown in Figure 2A, GMab-r1
recognized only IDH1-R132G protein expressed in CHO cells,
but not the other proteins (IDH1-WT and IDH1-R132L). LMab1 recognized only IDH1-R132L protein expressed in CHO cells,
not the other proteins (IDH1-WT and IDH1-R132G). These
results indicate that GMab-r1 and LMab-1 are useful in detecting IDH1-R132G or IDH1-R132L expressed in mammaliancells, respectively. As shown in Figure 2A, RcMab-1 strongly
recognized IDH1-WT, IDH1-R132G, and IDH1-R132L. RcMab1 also detects endogenous hamster IDH1.
Immunocytochemical analyses by anti-mutated IDH1
MAbs against mutated IDH1-expressing cell lines
Newly established anti-IDH1 MAbs GMab-r1 and LMab-1
were confirmed to be anti-mutated IDH1-specific antibodies
in Western blot analyses; therefore, we performed immunocytochemical (ICC) analyses using IDH1-R132G- or IDH1R132L-transfected CHO cells. Results show that GMab-r1
and LMab-1 reacted with IDH1-R132G- or IDH1-R132Ltransfected cells, respectively. RcMab-1 reacted with IDH1-

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227
In conclusion, the newly established anti-mutated IDH1specific MAbs GMab-r1 and LMab-1 are expected to be extremely useful for diagnosis and biological evaluation of
mutation-bearing gliomas in combination with previously
established anti-mutated IDH1-specific monoclonal antibodies (HMab-1 and SMab-1). Additional IDH1 mutationspecific antibodies such as anti-IDH1-R132C must be
developed for pathological diagnosis of gliomas, cartilaginous tumors, and AML.
Acknowledgments
We thank Shunpei Morita, Ryo Yanagiya, Kana Nasu,
Hiroko Sasaki, Sachiko Suzuki, Junko Aita for their excellent
technical assistance, and thanks to Dr. Taro Tachibana for his
help. 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.); by KAKENHI (23790185, 23701043): a Grant-in-Aid forYoung Scientists (M.K.K., Y.K.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; and by
the Intelligent Cosmos Academic Foundation (Y.K.).
Author Disclosure Statement
The authors have no financial interests to disclose.
References

FIG. 2. (A) 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) andIDH1 mutants (lane 2, IDH1-R132G; lane 3, IDH1-R132L)
were electrophoresed under reducing conditions using 5–
20% gels, and were Western blotted with GMab-r1, LMab-1,
RcMab-1, and anti-V5 tag. RcMab-1 detects two bands, including recombinant human IDH1 (upper band) and endogenous
hamster
IDH1(lowerband).
(B)
Immunocytochemical analyses by anti-IDH1 MAbs. CHO
cells were transfected by IDH1-R132G or IDH1-R132L. Eachcell was stained by GMab-r1, LMab-1, or RcMab-1, followedby rat IgG-Alexa 488 antibody (green). Cells were also treated with TO-PRO-3 to stain the cell nuclei (blue).
WT-expressing CHO cells, suggesting that RcMab-1 is also
useful in ICC. Next, we investigated the specificity of GMabr1 and LMab-1 against mutated IDH1-expressing CHO cells.
As depicted in Figure 2B, GMab-r1 reacted with only CHO/
IDH1-R132G, but not with CHO/IDH1-R132L. Immunostaining of GMab-r1 for CHO/IDH1-R132G demonstrated cytosolic patterns. In contrast, LMab-1 reacted with
only CHO/IDH1-R132L, not with CHO/IDH1-R132G. Immunostaining of LMab-1 for CHO/IDH1-R132L also demonstrated cytosolic patterns. These results indicate that
GMab-r1 and LMab-1 are useful in detecting IDH1-R132G or
IDH1-R132L, respectively, suggesting that GMab-r1 andLMab-1 could be also useful in immunohistochemical analyses for detection of IDH1-R132G or IDH1-R132L. In thesame way with Western blot analyses, RcMab-1 reacted withboth IDH1-R132G and IDH1-R132L (Fig. 2B).

1. Dang L, White DW, Gross S, Bennett BD, Bittinger MA,
Driggers EM, Fantin VR, Jang HG, Jin S, Keenan MC, Marks
KM, Prins RM, Ward PS, Yen KE, Liau LM, Rabinowitz JD,
Cantley LC, Thompson CB, Vander Heiden MG, and Su SM:
Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature 2009;462:739–744.
2. Struys EA, Salomons GS, Achouri Y, Van Schaftingen E,
Grosso S, Craigen WJ, Verhoeven NM, and Jakobs C: Mutations in the D-2-hydroxyglutarate dehydrogenase genecause D-2-hydroxyglutaric aciduria. Am J Hum Genet 2005;
76:358–360.
3. Kolker S, Pawlak V, Ahlemeyer B, Okun JG, Horster F,
Mayatepek E, Krieglstein J, Hoffmann GF, and Kohr G:
NMDA receptor activation and respiratory chain complex V
inhibition contribute to neurodegeneration in d-2-hydroxyglutaric aciduria. Eur J Neurosci 2002;16:21–28.
4. Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan
W, Kos I, Batinic-Haberle I, Jones S, Riggins GJ, Friedman H,Friedman A, Reardon D, Herndon J, Kinzler KW, Velculescu
VE, Vogelstein B, and Bigner DD: IDH1 and IDH2 mutationsin gliomas. N Engl J Med 2009;360:765–773.
5. Pansuriya TC, van Eijk R, d’Adamo P, van Ruler MA, Kuijjer
ML, Oosting J, Cleton-Jansen AM, van Oosterwijk JG, Verbeke SL, Meijer D, van Wezel T, Nord KH, Sangiorgi L,
Toker B, Liegl-Atzwanger B, San-Julian M, Sciot R, Limaye
N, Kindblom LG, Daugaard S, Godfraind C, Boon LM,
Vikkula M, Kurek KC, Szuhai K, French PJ, and Bovee JV:Somatic mosaic IDH1 and IDH2 mutations are associated withenchondroma and spindle cell hemangioma in Ollier disease
and Maffucci syndrome. Nat Genet 2011;43:1256–1261.
6. Amary MF, Damato S, Halai D, Eskandarpour M, Berisha F,
Bonar F, McCarthy S, Fantin VR, Straley KS, Lobo S, Aston
W, Green CL, Gale RE, Tirabosco R, Futreal A, Campbell P,
Presneau N, and Flanagan AM: Ollier disease and Maffucci
syndrome are caused by somatic mosaic mutations of IDH1
and IDH2. Nat Genet 2011;43:1262–1265.

228
7. Ward PS, Patel J, Wise DR, Abdel-Wahab O, Bennett BD,
Coller HA, Cross JR, Fantin VR, Hedvat CV, Perl AE, Rabinowitz JD, Carroll M, Su SM, Sharp KA, Levine RL, andThompson CB: The common feature of leukemia-associated
IDH1 and IDH2 mutations is a neomorphic enzyme activity
converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer Cell 2010;17:225–234.
8. Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, Angenendt
P, Mankoo P, Carter H, Siu IM, Gallia GL, Olivi A,
McLendon R, Rasheed BA, Keir S, Nikolskaya T, Nikolsky Y,
Busam DA, Tekleab H, Diaz LA Jr, Hartigan J, Smith DR,
Strausberg RL, Marie SK, Shinjo SM, Yan H, Riggins GJ,
Bigner DD, Karchin R, Papadopoulos N, Parmigiani G,
Vogelstein B, Velculescu VE, and Kinzler KW: An integrated
genomic analysis of human glioblastoma multiforme. Science 2008;321:1807–1812.
9. Sonoda Y, Kumabe T, Nakamura T, Saito R, Kanamori M,
Yamashita Y, Suzuki H, and Tominaga T: Analysis of IDH1
and IDH2 mutations in Japanese glioma patients. Cancer Sci
2009;100:1996–1998.
10. Balss J, Meyer J, Mueller W, Korshunov A, Hartmann C, andvon Deimling A: Analysis of the IDH1 codon 132 mutationin brain tumors. Acta Neuropathol 2008;116:597–602.
11. Hartmann C, Meyer J, Balss J, Capper D, Mueller W,
Christians A, Felsberg J, Wolter M, Mawrin C, Wick W,
Weller M, Herold-Mende C, Unterberg A, Jeuken JW, Wesseling P, Reifenberger G, and von Deimling A: Type andfrequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: astudy of 1,010 diffuse gliomas. Acta Neuropathol 2009;118:469–474.
12. von Deimling A, Korshunov A, and Hartmann C: The next
generation of glioma biomarkers: MGMT methylation, BRAF
fusions and IDH1 mutations. Brain Pathol 2011;21:74–87.
13. Takano S, Kato Y, Yamamoto T, Kaneko MK, Ishikawa E,
Tsujimoto Y, Matsuda M, Nakai K, Yanagiya R, Morita S,
Tsuboi K, and Matsumura A: Immunohistochemical detection of IDH1 mutation, p53, and internexin as prognostic
factors of glial tumors. J Neurooncol 2012;108:361–373.
14. Takano S, Tian W, Matsuda M, Yamamoto T, Ishikawa E,
Kaneko MK, Yamazaki K, Kato Y, and Matsumura A: Detection of IDH1 mutation in human gliomas: comparison of

KANEKO ET AL.
immunohistochemistry and sequencing. Brain Tumor Pathol
2011;28:115–123.
15. Kaneko MK, Tian W, Takano S, Suzuki H, Sawa Y, Hozumi
Y, Goto K, Yamazaki K, Kitanaka C, and Kato Y: Establishment of a novel monoclonal antibody SMab-1 specific for
IDH1-R132S mutation. Biochem Biophys Res Commun
2011;406:608–613.
16. Kato Y, Jin G, Kuan CT, McLendon RE, Yan H, and Bigner
DD: A monoclonal antibody IMab-1 specifically recognizes
IDH1R132H, the most common glioma-derived mutation.
Biochem Biophys Res Commun 2009;390:547–551.
17. Capper D, Zentgraf H, Balss J, Hartmann C, and vonDeimling A: Monoclonal antibody specific for IDH1 R132H
mutation. Acta Neuropathol 2009;118:599–601.
18. Sakaguchi N, Miyamoto Y, Yoneda Y, Ogino K, and Tachibana T: Generation of a rat monoclonal antibody specific forimportin alpha3/Qip1. Hybrid Hybridomics 2003;22:397–400.
19. Kaneko MK, Morita S, Tsujimoto Y, Yanagiya R, Nasu K,
Sasaki H, Hozumi Y, Goto K, Natsume A, Watanabe M, Kumabe T, Takano S, and Kato Y: Establishment of novel
monoclonal antibodies KMab-1 and MMab-1 specific for IDH2
mutations. Biochem Biophys Res Commun 2013;432:40–45.
20. Kato Y, Natsume A, and Kaneko MK: A novel monoclonal
antibody GMab-m1 specifically recognizes IDH1-R132G mutation. Biochem Biophys Res Commun 2013;432(4):564–567.
21. Kato Y and Kaneko MK: Generation of a novel monoclonal
antibody WMab-1 specific for IDH2-R172W mutation. Biochem Biophys Res Commun 2013;433:374–378.

Address correspondence to:
Dr. Yukinari Kato
Tohoku University
Graduate School of Medicine
2-1 Seiryo-machi
Aoba-ku, Sendai
Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp
Received: January 20, 2013
Accepted: February 27, 2013