MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 35, Number 5, 2016ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2016.0037

Establishment of Anti-Human ATRX
Monoclonal Antibody AMab-6
Satoshi Ogasawara,1 Yuki Fujii,1 Mika K. Kaneko,1 Hiroharu Oki,1 Hemragul Sabit,2
Mitsutoshi Nakada,2 Hiroyoshi Suzuki,3 Koichi Ichimura,4 Takashi Komori,5 and Yukinari Kato1

Gliomas are the most frequently occurring brain tumors with a heterogeneous molecular background. The molecularsubgrouping of gliomas more prognostically stratifies patients into distinct groups compared with conventional
histological classification. The most important molecules for the subtype diagnosis of diffuse gliomas are mutationsof isocitrate dehydrogenase (IDH), TERT promoter, and a-thalassemia/mental-retardation-syndrome-X-linked(ATRX) and the codeletion of 1p/19q. Among them, IDH and ATRX mutations can be diagnosed using specificmonoclonal antibodies (mAbs). We have developed many mAbs against IDH mutants, including HMab-1/HMab-2
against IDH1-R132H and multispecific mAbs MsMab-1/MsMab-2 against IDH1/2 mutations. In contrast, highly
sensitive mAbs against ATRX remain to be established. In this study, we immunized mice with recombinant human
ATRX and developed a novel mAb, AMab-6. The dissociation constant of AMab-6 was determined to be 9.7 · 10-10
M, indicating that the binding affinity of AMab-6 is very high. Furthermore, AMab-6 sensitively detects ATRX in
Western blot and immunohistochemical analyses, indicating that AMab-6 could become the standard marker todetermine the ATRX mutation status of gliomas in immunohistochemical analyses.
Keywords: ATRX, glioma, immunohistochemistry

IntroductionT

he molecular subgrouping of gliomas using mutations of isocitrate dehydrogenase (IDH), a-thalassemia/
mental-retardation-syndrome-X-linked (ATRX), and TERT
promoter and the status of 1p/19q more prognosticallystratify patients into distinct groups compared with conventional histological classification.(1–4) Treatment strategies
can be planned with respect to molecular subtype along with
the WHO grade; therefore, these molecular subtypes areclinically important. The inclusion of molecular parametersin the WHO definition of brain tumors was forwarded as the‘‘ISN-Haarlem’’ consensus.(5) The 2016 WHO Classificationof Tumors of the Central Nervous System (2016 CNS WHO)is both a conceptual and practical advance over the 2007 CNS
WHO. The 2016 CNS WHO uses molecular parameters in
addition to histology to define many tumor entities; presents
major restructuring of the diffuse gliomas, medulloblastomas, and other embryonal tumors; and incorporates newentities, which are defined by both histology and molecularfeatures.(6)ATRX mutation in gliomas results in the loss of ATRX
protein expression, which can be diagnosed by immunohistochemistry using anti-ATRX antibodies.(7) Almost all studies of
ATRX have used polyclonal antibodies(8) because highly
sensitive monoclonal antibodies (mAbs) against human ATRX
remain to be established. Herein, we report a novel anti-ATRX
mAb, AMab-6, which is very useful in enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemical analyses.
Materials and Methods
Cell lines and animals

P3 U1 was obtained from the American Type Culture
Collection (Manassas, VA) and was cultured in the RPMI
1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented with 10% heat-inactivated fetal bovine serum(Thermo Fisher Scientific, Inc., Waltham, MA) at 37C in ahumidified atmosphere of 5% CO2 and 95% air. Antibiotics,
including 100 U/mL of penicillin, 100 mg/mL of streptomycin, and 25 mg/mL of amphotericin B (Nacalai Tesque, Inc.),1

Department of Regional Innovation, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Neurosurgery, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan.
3
Department of Pathology and Laboratory Medicine, Sendai Medical Center, Sendai, Japan.
4
Division of Brain Tumor Translational Research, National Cancer Center Research Institute, Tokyo, Japan.
5
Department of Neuropathology, Tokyo Metropolitan Neurological Hospital, Tokyo, Japan.
2254

ANTI-HUMAN ATRX MAB

were added to the media. Female MRL/lpr mice (4 weeksold) were purchased from SLC Japan (Shizuoka, Japan).
Animals were housed under pathogen-free conditions. The‘‘Animal Care and Use Committee of Tohoku University’’approved the animal experiments described herein.
Hybridoma production

Plasmid preparation and ATRX recombinant protein production are described in the Supplementary Data. MRL/lpr
mice were immunized using an intraperitoneal (i.p.) injection of PA-ATRX-RAP-MAP (100 mg) together with Imject
Alum (Thermo Fisher Scientific, Inc.). After three additional
immunizations, a booster injection was administered byi.p. 2 days before the spleen cells were harvested. The spleencells were fused with P3 U1 cells using PEG1500 (Roche
Diagnostics, Indianapolis, IN). The fused cells were grown in
the RPMI medium with hypoxanthine, aminopterin, andthymidine selection medium supplement (Thermo Fisher
Scientific, Inc.). Culture supernatants were screened using
ELISA for the binding to recombinant MBP-ATRX-MAPPA or GST-ATRX-MAP-PA.

255
Immunohistochemical analysis

Surgical specimens of oligodendroglioma and diffuse astrocytoma were fixed in 4% paraformaldehyde and embedded
in paraffin for routine histopathological and immunohistochemical analysis. Immunohistochemical analysis was performed using avidin-biotin immunoperoxidase technique. In
brief, 4-mm-thick tissue sections were deparaffinized using
xylene and rehydrated. After antigen retrieval using Target
Retrieval Solution, pH 9.0 (Dako), the sections were autoclavedfor 10 minutes and then maintained at room temperature for 40
minutes. Sections were quenched using 3% hydrogen peroxide(H2O2) in methanol for 20 minutes and blocked with 5% skim
milk in TBST for 30 minutes. Sections were incubated with
3 mg/mL AMab-6 overnight at 4C. Immunocomplexes weretreated with an Envision+ Kit (Dako) for 1 hour. Color wasdeveloped using 3, 3-diaminobenzidine tetrahydrochloride(DAB Substrate Kit SK-4100, Vector, Japan) for 5 minutes.
Sections were then counterstained with hematoxylin (Wako
Pure Chemical Industries Ltd.). Images were acquired with a
BZ-X700 microscope (Keyence, Osaka, Japan).
Results and Discussion

Enzyme-linked immunosorbent assay

Recombinant MBP-ATRX-MAP-PA or GST-ATRXMAP-PA was immobilized on Nunc MaxiSorp 96-well immunoplates (Thermo Fisher Scientific, Inc.) at a concentration of 5 mg/mL for 30 minutes. After blocking with 1%
bovine serum albumin in 0.05% Tween 20/phosphatebuffered saline, the plates were incubated with culture supernatant followed by 1:3000-diluted peroxidase-conjugatedanti-mouse IgG (Dako; Agilent Technologies, Inc., Glostrup,
Denmark). The enzymatic reaction was performed with a 1Step Ultra TMB-ELISA (Thermo Fisher Scientific, Inc.). The
optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA).

The two major methods for identifying these subtypes ofgliomas are direct DNA sequencing and immunohistochemical analyses using antibodies.(9) Immunohistochemistry is a

Determination of binding affinity using ELISA

Purified recombinant protein was immobilized at 5 mg/mL.
The plates were incubated with serially diluted antibodies(26 pg/mL–10 mg/mL) followed by 1:1000-diluted peroxidaseconjugated anti-mouse IgG (Dako). The dissociation constant(KD) was obtained by fitting the binding isotherms using thebuilt-in one-site binding models in GraphPad Prism 6(GraphPad Software, Inc., La Jolla, CA).
Western blot analyses

Recombinant protein (0.1 mg) was boiled in SDS sample
buffer (Nacalai Tesque, Inc.). The proteins were electrophoresed on 5%–20% polyacrylamide gels (Wako Pure
Chemical Industries Ltd., Osaka, Japan) and were transferred
onto a PVDF membrane (EMD Millipore Corp., Billerica,
MA). After blocking with 4% skim milk (Nacalai Tesque,Inc.), the membrane was incubated with AMab-6 or antiMBP (clone: TMab-2; Wako Pure Chemical Industries Ltd.)and then with peroxidase-conjugated anti-mouse or anti-rat
antibodies (1:1000 diluted; Dako) and developed with the
Pierce Western Blotting Substrate Plus (Thermo Fisher Scientific, Inc.) or the ImmunoStar LD Chemiluminescence
Reagent (Wako Pure Chemical Industries Ltd.) using the
Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).

FIG. 1. Purification of the recombinant ATRX protein.
Cleared lysate (5 mL) from the transformed Escherichia coli
before (input) and after (flow through) NZ-1-Sepharose
capture, as well as 10 elution fractions (lanes 1–10) during
column chromatography, was subjected to 5%–20% gradient SDS-PAGE under a reducing condition and stained with
CBB. ATRX, a-thalassemia/mental-retardation-syndromeX-linked; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

256

robust and widely available method, and immunohistochemical approaches with defined protocols and materials
have become essential for assessing molecular genetic
changes.(10) Among many molecular parameters, the important molecules for subtype diagnosis of diffuse gliomas are
mutations of IDH, TERT promoter, and ATRX and the codeletion of 1p/19q.(11) In particular, mutations of IDH(9) andATRX(7,8) can be accurately detected by immunohistochemical analysis. We developed many mAbs against IDH
mutants, including HMab-1/HMab-2 against IDH1-R132H
and multispecific mAbs MsMab-1/MsMab-2 against IDH1/2
mutations.(7,9,12–28) However, highly sensitive mAbs against
human ATRX remain to be established.
In this study, the recombinant PA-ATRX-RAP-MAP was
captured using NZ-1-Sepharose and efficiently eluted with
MES buffer (pH 6.0), containing 3 M MgCl2 (Fig. 1). Mice
were then immunized with PA-ATRX-RAP-MAP to develop
novel anti-ATRX mAbs. We utilized the C-terminal region of
ATRX as immunogen because commercially available polyclonal antibodies (Sigma-Aldrich) against ATRX were also
produced against that region.(7) Using ELISA, the culture supernatants of hybridomas were screened for binding to recombinant MBP-ATRX-MAP-PA or GST-ATRX-MAP-PA
that was purified from Escherichia coli. As a result, AMab-6(mouse IgG1, kappa) was established after limiting dilution.
As shown in Figure 2, AMab-6 reacted with MBP-ATRXMAP-PA in a dose-dependent manner. In contrast, AMab-6
did not react with MBP-IDH1. We further performed akinetic analysis of the interaction of AMab-6 with MBP-ATRX-MAP-PA (Fig. 2). The KD of AMab-6 was determined to be 9.7 · 10-10 M, indicating that the apparent
binding affinity of AMab-6 is very high.
Western blot analysis demonstrated that AMab-6 detected
MBP-ATRX-MAP-PA, but not MBP-IDH1, indicating that
AMab-6 does not detect MBP tag in Western blot analysis(Fig. 3). Immunohistochemical analyses revealed that AMab-6
reacted with the nuclei of oligodendroglioma cells without
ATRX mutations (Fig. 4A). ATRX mutations in gliomas result

FIG. 2. Determination of binding affinity using enzymelinked immunosorbent assay. Purified recombinant protein(MBP-ATRX-MAP-PA or MBP-IDH1) was immobilized at
5 mg/mL. The plates were incubated with serially diluted
antibodies (26 pg/mL–10 mg/mL) followed by 1:1000-diluted
peroxidase-conjugated anti-mouse IgG. The dissociation
constant (KD) was obtained by fitting the binding isotherms
using the built-in one-site binding models in GraphPad Prism
6. IDH, isocitrate dehydrogenase.

OGASAWARA ET AL.

FIG. 3. Western blot analysis. Recombinant proteins(0.1 mg) of MBP-ATRX-MAP-PA and MBP-IDH1 were
electrophoresed and transferred onto a PVDF membrane.
After blocking, the membrane was incubated with AMab-6,
RMab-3 (anti-IDH1), or TMab-2 (anti-MBP).

FIG. 4. Immunohistochemical analysis. (A) Oligodendroglioma, IDH-mutant (WHO grade II). Note the ATRX
immunostaining in the nucleus of tumor cells without
ATRX mutation. (B) Diffuse astrocytoma, IDH mutant(WHO grade II). Note the negative staining of ATRX in the
nucleus of tumor cells with ATRX mutation, whereas positive staining was observed in the vascular endothelial cells
without ATRX mutation.

ANTI-HUMAN ATRX MAB

in the loss of ATRX protein expression, which can be diagnosed
using immunohistochemical analysis.(7) Likewise, AMab-6 did
not recognize the nuclei of diffuse astrocytoma cells, which
possess ATRX mutation, but reacted with those of vascularendothelial cells without ATRX mutation (Fig. 4B). These results indicate that AMab-6 is very sensitive against ATRX,
which is expressed in gliomas without ATRX mutation.
In conclusion, a novel anti-ATRX mAb, AMab-6, sensitively reacted with human ATRX in ELISA, Western blot,
and immunohistochemical analyses. Although many studies
have used commercially available polyclonal anti-ATRX
antibodies,(7) we confirmed that the reactivity of AMab-6 iscompatible with that of polyclonal antibodies (data not shown).
Furthermore, we performed immunohistochemical analyses
under many other conditions for antigen retrievals (citratebuffer, pH 6.0 or 9.0) or incubation time of AMab-6 (30 minutes, 1 hour, or overnight) and obtained positive staining results
as shown in Figure 4. Altogether, AMab-6 could serve as astandard marker to determine the ATRX mutation status ofgliomas during immunohistochemical analyses.
Acknowledgments

The authors thank Takuro Nakamura, Noriko Saidoh, andKanae Yoshida for their excellent technical assistance. This
work was supported, in part, by the Practical Research for
Innovative Cancer Control from Japan Agency for Medical Research and development, AMED (Y.K.); by JSPS
KAKENHI Grant Number 25462242 (Y.K.) and 16K10748(Y.K.), by the Regional Innovation Strategy Support Programfrom the Ministry of Education, Culture, Sports, Science andTechnology (MEXT) of Japan (Y.K.), by the Platform for Drug
Discovery, Informatics, and Structural Life Science (PDIS)
from AMED (Y.K.), and by the Basic Science and Platform
Technology Program for Innovative Biological Medicine from
AMED (Y.K.). This work was performed, in part, under the
Cooperative Research Program of Institute for Protein Research, Osaka University, CR-15-05 and CR-16-05.
Author Disclosure Statement

No competing financial interests exist.
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Address correspondence to:
Dr. Yukinari Kato
Department of Regional Innovation
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;
yukinarikato@med.tohoku.ac.jp
Received: September 6, 2016
Accepted: September 15, 2016