Biochemical and Biophysical Research Communications 391 (2010) 750–755

Contents lists available at ScienceDirect

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

GMab-1, a high-affinity anti-30 -isoLM1/30 ,60 -isoLD1 IgG monoclonal antibody,raised in lacto-series ganglioside-defective knockout mice
Yukinari Kato a,b,*, Chien-Tsun Kuan a, Jinli Chang a, Mika Kato Kaneko a, Joanne Ayriss a, Hailan Piao a,
Vidyalakshmi Chandramohan a, Charles Pegram a, Roger E. McLendon a, Pam Fredman c, Jan-Eric Månsson c,Darell D. Bigner aaDepartment 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, Japanc
Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgren Academy at University of Gothenburg, Sahlgren University Hospital/Molndal,
SE-431 80 Molndal, Swedenb

a r t i c l e

i n f oArticle history:
Received 16 November 2009
Available online 26 November 2009
Keywords:
Lacto-series ganglioside
30 -IsoLM1
30 ,60 -IsoLD1
Monoclonal antibody
Lc3-synthase
Knockout micea b s t r a c t
The lacto-series gangliosides 30 -isoLM1 and 30 ,60 -isoLD1 have been identified as tumor-associated antigens whose formation is initiated by the Lc3-synthase. Until now, high-affinity IgG monoclonal antibodies (mAbs) against 30 -isoLM1 and 30 ,60 -isoLD1, which are highly expressed in gliomas, have not beendeveloped, although mAbs against lacto-series gangliosides are powerful tools for functional studies.
We previously produced the Lc3-synthase gene b3Gn-T5 knockout mice. In this study, we immunizedb3Gn-T5 knockout mice with 30 -isoLM1/30 ,60 -isoLD1 and produced the anti-30 -isoLM1/30 ,60 -isoLD1 mAb
GMab-1, of the IgG3 subclass, which should be useful for functional analysis of lacto-series gangliosides
and for antibody-based therapy of gliomas.
! 2009 Elsevier Inc. All rights reserved.

Introduction
Gangliosides are sialic acid-containing glycosphingolipids
highly enriched in the vertebrate nervous system. Although theirfunctions have not been fully clarified, they are thought to mediateneural cell–cell recognition and modulate intracellular signaling[1]. Gangliosides are implicated in various neural disorders, primarily the autoimmune neuropathies, but also in storage disorders, e.g.,
Tay–Sachs disease or Sandhoff disease [2–4]. High-affinity IgG antiganglioside antibodies may be used to develop animal models of
autoimmune neuropathies and probe normal ganglioside functions.
However, it has been difficult to produce high-affinity IgG antibodies against major brain gangliosides, especially GM1, GD1a, and GT
1b [5]. This unresponsiveness has been attributed to poor immunogenicity, T-cell independence, and tolerance [5–7]. Advances ingenetics provide a potential solution to this problem. Several studies have shown that mice genetically engineered to lack the glycosyltransferase gene for ganglioside synthesis do not express
Abbreviations: b3Gn-T5, b1,3-N-acetylglucosaminyltransferase-V; BSA, bovine
serum albumin; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine
serum; KA, affinity constant; kassoc, association rate constant; kdiss, disassociation
rate constant; mAb, monoclonal antibody* 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 ! 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2009.11.132

complex gangliosides [8–12]. These mice, immunologically naive
to complex gangliosides, have been used for raising new antibodies
against complex gangliosides [2,6,13,14].
The Lc3-synthase gene (b1,3-N-acetylglucosaminyltransferaseV: b3Gn-T5) enzyme initiates the formation of the lacto-/neolacto-series ganglioside by transferring GlcNAc in a b1,3-linkage tolactosylceramide (Fig. 1A) [15]. The b3Gn-T5 is detected in mouse
development and then again later mainly in the spleen and placenta in adult mice. Additionally, Lc3-synthase transcripts arefound in cerebellar Purkinje cells of the adult mouse brain. On
the other hand, lacto-series gangliosides such as 30 -isoLM1 and30 ,60 -isoLD1 have been reported to be major mono- and oligo-sialogangliosides, respectively, of human gliomas [16–18]. In thosestudies, monoclonal antibodies (mAbs) such as SL-50, DMab-14,
or DMab-22 recognizing lacto-series gangliosides were successfully produced; however, those antibodies proved to be of the
low-affinity IgM subclass. In this study, we immunized b3Gn-T5
knockout mice with purified 30 -isoLM1 and 30 ,60 -isoLD1 to overcome these problems and generated an anti-lacto-series ganglioside IgG antibody with high affinity.
Materials and methods
Animals, cell lines, xenograft, and tissues. The b3Gn-T5 knockout
mice were previously developed at Duke University Medical

Y. Kato et al. / Biochemical and Biophysical Research Communications 391 (2010) 750–755

751

Fig. 1. Production of an anti-30 -isoLM1/30 ,60 -isoLD1 ganglioside antibody. (A) Biosynthesis of lacto-/neolacto-series ganglioside. (B) Electrophoresis of 2 lg of GMab-1 under
reducing conditions on 4–10% NuPAGE gel. (C) ELISA of GMab-1 against 30 -isoLM1. The 30 -isoLM1 conjugated with BSA was immobilized. After blocking, the plates wereincubated with GMab-1 and isotype control at several concentrations.

Center. P3U1 cells were obtained from the American Type Culture
Collection (Manassas, VA), and we established a D54MG glioblastoma cell line at Duke [16]. P3U1 and D54MG cells 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) and 1% of penicillin–streptomycin solution (Invitrogen Corp.) or Zinc Option medium supplemented with 10% heatinactivated fetal bovine serum (FBS; Sigma, St. Louis, MO), respectively. We established and maintained a D54MG xenograft at Duke,derived from cultured D54MG cells. Human tissues slides fromanonymous donors were obtained from the Tissue Bank at the
Preston Robert Tisch Brain Tumor Center at Duke University.
Antibodies and gangliosides. Anti-ganglioside antibodies SL-50,
DMab-14, and DMab-22 were previously produced at Duke andthe University of Gothenburg [16–18]. Isotype control of mouse
IgG3 was purchased from eBioscience, Inc. (San Diego, CA). All gan-gliosides used for immunization or enzyme-linked immunosorbent
assay (ELISA) were isolated and characterized at the University ofGothenburg as described previously [16,17,19].
Hybridoma production. The b3Gn-T5 knockout mice were immunized by neck s.c. injections of 20 lg of purified 30 -isoLM1 and30 ,60 -isoLD1 coupled to Salmonella minnesota with Imject Freund’sComplete Adjuvant (Thermo Scientific Inc., Rockford, IL). One week
later, secondary i.p. immunization of 20 lg of purified gangliosides
was performed. After additional immunization of 20 lg of purified
gangliosides, a booster injection was given i.p. 2 days before spleencells were harvested. The spleen cells were fused with mouse myeloma P3U1 cells by using Sendai virus (hemagglutinating virus ofJapan: HVJ) envelope: GenomONE-CFEX (Cosmo Bio USA, Inc., Carlsbad, CA) according to the manufacturer’s instructions. The hybridomas were grown in RPMI medium including hypoxanthine,aminopterin, and thymidine selection medium supplement

752

Y. Kato et al. / Biochemical and Biophysical Research Communications 391 (2010) 750–755(Sigma), 2 mM L-glutamine (Invitrogen Corp.), 10% heat-inactivatedFBS (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 the binding
to 30 -isoLM1 conjugated with fatty-acid free-bovine serum albumin (BSA). Single cell cloning was performed with ClonaCell-HY
Hybridoma Selection Medium (Medium D; StemCell Technologies
Inc., Vancouver, BC, Canada). The IgG subclass was determined by
IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics Corp., Indianapolis, IN).
Enzyme-linked immunosorbent assay (ELISA). For evaluation by
ELISA, gangliosides conjugated with BSA were immobilized on
96-well plates at 1 lg/ml for 30 min. After blocking with 1% BSA
in PBS, the plates were incubated with primary antibodies at several concentrations, followed by 1:1000 diluted horseradish-peroxidase-conjugated anti-mouse IgG (GE Healthcare UK Ltd.,
Buckinghamshire, England). The enzymatic reaction was conducted with a substrate solution containing 3,30 ,5,50 -tetramethylbenzidine (TMB; Thermo Scientific Inc.). After the reaction was
stopped with 2 M H2SO4, the optical density was measured at
450 nm with a microplate reader (Bio-Rad Laboratories, Inc., Philadelphia, PA). These reactions were performed with a volume of
50 ll at room temperature.
SDS–PAGE. For sodium dodecyl sulfate polyacrylamide gel electrophoresis, 2 lg of each protein was electrophoresed under reducing conditions on 4–10% NuPAGE gel (Invitrogen). The gel was
stained with Bio-Safe Coomassie (Bio-Rad Laboratories, Inc.).
Flow cytometry. D54MG or D54MG xenograft cells, which werecollected by 0.6 mM EDTA treatment, were incubated with
GMab-1 or isotype control (10 lg/ml) for 1 h at 4 "C. Then the cells
were incubated with the Oregon green-conjugated anti-mouse
antibody (1/200 dilution; Invitrogen Corp.) for 30 min. Flowcytometry was performed with a FACS Calibur instrument (Becton
Dickinson, Franklin Lakes, NJ).
Affinity constant determination by surface plasmon resonance. To
determine the affinity constant (KA), purified GMab-1 was immobilized on the surface of biosensor chips for analysis by using the
BIAcore 3000 system (BIAcore, Piscataway, NJ). Coupling of
GMab-1 was achieved by using N-ethyl-N0 -(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide according to the instructions of the manufacturer. The running buffer was 10 nM HEPES,
150 mM NaCl, and 3.4 mM EDTA (pH 7.4). The gangliosides conjugated with BSA and control BSA were passed over the biosensor
chip and affinity rate constants (association rate constant, kassoc,

and disassociation rate constant, kdiss) were determined by nonlinear curve-fitting using the Langmuir one-site binding model of the
BIAevaluation software (BIAcore). KA at equilibrium was calculated
as KA = kassoc/kdiss.
Immunohistochemical analysis. All procedures were performed
with the Ventana Discovery XT system (Ventana Medical Systems,
Inc., Tucson, AZ). Briefly, acetone-fixed 5- to 8-lm frozen tissue
sections of human glioblastoma tissues were incubated with
GMab-1 (10 lg/ml) or isotype control (10 lg/ml) for 30 min. The
universal biotin-conjugated secondary antibody was incubatedwith tissue sections for 20 min followed by the horseradish-peroxidase-conjugated streptavidin for 16 min. Color was developed byusing 3,30 -diaminobenzidine tetrahydrochloride for 8 min, andhematoxylin was used for counterstained.
Results and discussion
Production of an anti-30 -isoLM1/30 ,60 -isoLD1-specific antibody
Monoclonal antibodies recognizing lacto-series gangliosides
such as 30 -isoLM1 and 30 ,60 -isoLD1 were previously produced andfully characterized [16,17]. The 30 -isoLM1 and 30 ,60 -isoLD1 gangliosides are highly expressed in human glioblastomas and are rarely
detected in the normal adult brain. Therefore, 30 -isoLM1 and 30 ,60 isoLD1 are ideal molecular targets for immunotherapy against glioblastomas using specific mAbs. However, those established mAbs
have relatively low-affinity against 30 -isoLM1 and 30 ,60 -isoLD1 because they are of the IgM subclass. In several studies, anti-ganglioseries ganglioside IgGs with high affinity have been developed byimmunizing GalNAcT!/! mice with ganglio-series gangliosides[2,6,13,14]. Similarly, in this study, we immunized b3Gn-T5 knockout mice with purified 30 -isoLM1 and 30 ,60 -isoLD1 coupled to S. minnesota to generate high-affinity anti-lacto-series ganglioside mAbs
(Fig. 1A). Because the hybridomas that we previously produced fromb3Gn-T5 knockout mice using the conventional polyethylene glycol
fusion method had low viability (data not shown), for this study, we
selected a low-toxicity, high-efficiency fusion method using a Sendai virus envelope [20]. After single cell cloning, one of the clones,
GMab-1 (IgG3 subclass) was established (Fig. 1B). As shown in
Fig. 1C, GMab-1 reacted with 30 -isoLM1 in a dose-dependent manner
in ELISA. Furthermore, both D54MG cells and D54MG xenograft
cells, which express lacto-series gangliosides, were recognized by
GMab-1 as demonstrated by flow cytometry (Fig. 2). To determine
the association and dissociation rate constants (kassoc and kdiss) and

Fig. 2. GMab-1 recognition of lacto-series ganglioside on the cell surface of glioblastoma cells by flow cytometry. D54MG cell line (A) or D54MG xenograft cells (B), whichwere collected by 0.6 mM EDTA treatment, were incubated with GMab-1 or isotype control.

Y. Kato et al. / Biochemical and Biophysical Research Communications 391 (2010) 750–755

to calculate the affinity constants (KA), we next performed a kinetic
analysis of the interaction of GMab-1 with 30 -isoLM1 by surface
plasmon resonance (BIAcore). Determination of the associationand dissociation rates from the sensorgrams revealed a kassoc of
5.07 " 104 (mol/l s)!1 and a kdiss of 2.74 " 10!4 s!1. The KA at binding equilibrium, calculated as KA = kassoc/kdiss, was 1.85 " 108 (mol/l)!1, indicating that GMab-1 has high affinity against 30 -isoLM1.
Likewise, the KA of the interaction of GMab-1 with 30 ,60 -isoLD1
was calculated as 6.58 " 107 (mol/l)!1, indicating that GMab-1 also
has high affinity against 30 ,60 -isoLD1.
Specificity of GMab-1 against gangliosides
To determine the epitope of GMab-1, we performed ELISA using
15 different gangliosides, including lacto-series (30 -isoLM1, 30 ,60 isoLD1, isoLA1, and Fuc-30 -isoLM1), neolacto-series (30 -LM1 and30 ,8’-LD1), globo-series (Gb3), asilalo-series (GA1), a-series (GM2and GM1), and b-series (GD3, GD2, GD1b, and GT 1b) (Fig. 3). As

753

previously reported [16,17], SL-50 reacted with 30 -isoLM1 andnot with 30 ,60 -isoLD1 (Fig. 3C), whereas DMab-22 reacted with
30 ,60 -isoLD1 and not with 30 -isoLM1 (Fig. 3D). We have observed
no cross-reaction with the other gangliosides using SL-50 and
DMab-22. DMab-14 reacted with both 30 -isoLM1 and 30 ,60 -isoLD1;
however, there is slight reactivity with the other gangliosides(Fig. 3B). On the other hand, we did observe activity that distinguished GMab-1 from SL-50, DMab-22, and DMab-14. GMab-1 recognized both 30 -isoLM1 and 30 ,60 -isoLD1, and the reactivity with
30 ,60 -isoLD1 was stronger than with 30 -isoLM1 (Fig. 3A). Although
DMab-14 reacted with both 30 -isoLM1 and 30 ,60 -isoLD1, the reactivity with 30 -isoLM1 was stronger than with 30 ,60 -isoLD1(Fig. 3B), which indicates that the epitope of GMab-1 is slightly different from that of DMab-14. Furthermore, there is no cross-reaction with the other gangliosides using GMab-1 (Fig. 3A). These
results indicate that GMab-1 defines the epitope NeuAca23Galb1-3GlcNAc, the terminal sequence of 30 -isoLM1 and 30 ,60 isoLD1.

Fig. 3. Epitope determination of GMab-1 in ELISA. Fifteen gangliosides conjugated with BSA were immobilized. After blocking, the plates were incubated with GMab-1 (A),DMab-14 (B), SL-50 (C), and DMab-22 (D) at a concentration of 10 lg/ml.

754

Y. Kato et al. / Biochemical and Biophysical Research Communications 391 (2010) 750–755

Fig. 4. Immunohistochemical analysis by GMab-1 against glioblastoma tissues. Glioblastoma tissues were stained by GMab-1 (A,C) or isotype control (B,D). Magnification:
200".

Immunohistochemical analysis by GMab-1 against glioblastoma
tissues
Since lacto-series gangliosides such as 30 -isoLM1 and 30 ,60 isoLD1 are known to be expressed in glioblastoma tissues [18],immunohistochemistry was performed using 33 glioblastoma frozen tissue specimens. GMab-1 immunoreactivity was detected in
26 of 33 (78.8%) glioblastomas. Representative staining by GMab1 in glioblastoma samples is shown in Fig. 4. Immunostaining by
GMab-1 demonstrated predominantly both cell-surface patterns(Fig. 4A) and cell-surface/cytoplasmic patterns (Fig. 4C) in glioblastoma cells. Proliferating endothelial cells were negative for GMab1 (Fig. 4A). The concentration-matched isotype control of mouse
IgG3 did not stain any tumor cells (Fig. 4B and D). These results further suggest that GMab-1 specifically reacted with lacto-series
gangliosides in glioblastoma tissues.
In conclusion, we immunized b3Gn-T5 knockout mice with 30 isoLM1 and 30 ,60 -isoLD1. GMab-1, of the IgG3 subclass, specificallyrecognized both 30 -isoLM1 and 30 ,60 -isoLD1 and showed high binding activity. GMab-1 was also reactive against glioblastomas inimmunohistochemical analyses, which indicates that GMab-1
should be useful in antibody-based therapy of glioblastomas.
Acknowledgments
We thank Mr. Ling Wang of Duke University Medical Center andMrs. Birgitta Dellheden of University of Gothenburg for immunohistochemical analysis and ganglioside isolation, respectively. We
also thank Dr. Chifumi Kitanaka of Yamagata University School of
Medicine for his helpful suggestion and Ms. Janet Parsons of Duke
University Medical Center for editing the manuscript. This study
was supported by NIH Grants 5P50 CA108786, 5P50 NS20023,
and R37 CA011898. Y.K. was supported by the Global COE Programfor Medical Sciences, Japan Society for the Promotion of Science.
References[1] S.I. Hakomori, Structure and function of glycosphingolipids and sphingolipids:
recollections and future trends, Biochim. Biophys. Acta 1780 (2008) 325–346.

[2] T. Bowes, E.R. Wagner, J. Boffey, D. Nicholl, L. Cochrane, M. Benboubetra, J.
Conner, K. Furukawa, K. Furukawa, H.J. Willison, Tolerance to self gangliosides
is the major factor restricting the antibody response to lipopolysaccharide coreoligosaccharides in Campylobacter jejuni strains associated with Guillain–Barre
syndrome, Infect. Immun. 70 (2002) 5008–5018.
[3] H. Akeboshi, Y. Chiba, Y. Kasahara, M. Takashiba, Y. Takaoka, M. Ohsawa, Y.
Tajima, I. Kawashima, D. Tsuji, K. Itoh, H. Sakuraba, Y. Jigami, Production ofrecombinant beta-hexosaminidase A, a potential enzyme for replacement
therapy for Tay–Sachs and Sandhoff diseases, in the methylotrophic yeast
Ogataea minuta, Appl. Environ. Microbiol. 73 (2007) 4805–4812.
[4] N. Kawashima, D. Tsuji, T. Okuda, K. Itoh, K.I. Nakayama, Mechanism ofabnormal growth in astrocytes derived from a mouse model of GM2
gangliosidosis, J. Neurochem. 111 (2009) 1031–1041.
[5] I. Kawashima, O. Nakamura, T. Tai, Antibody responses to ganglio-series
gangliosides in different strains of inbred mice, Mol. Immunol. 29 (1992) 625–
632.
[6] M.P. Lunn, L.A. Johnson, S.E. Fromholt, S. Itonori, J. Huang, A.A. Vyas, J.E.
Hildreth, J.W. Griffin, R.L. Schnaar, K.A. Sheikh, High-affinity anti-ganglioside
IgG antibodies raised in complex ganglioside knockout mice: reexamination of
GD1a immunolocalization, J. Neurochem. 75 (2000) 404–412.
[7] H. Ozawa, M. Kotani, I. Kawashima, T. Tai, Generation of one set of monoclonal
antibodies specific for b-pathway ganglio-series gangliosides, Biochim.
Biophys. Acta 1123 (1992) 184–190.
[8] Y. Liu, R. Wada, H. Kawai, K. Sango, C. Deng, T. Tai, M.P. McDonald, K. Araujo,
J.N. Crawley, U. Bierfreund, K. Sandhoff, K. Suzuki, R.L. Proia, A genetic model ofsubstrate deprivation therapy for a glycosphingolipid storage disorder, J. Clin.
Invest. 103 (1999) 497–505.
[9] K. Takamiya, A. Yamamoto, K. Furukawa, S. Yamashiro, M. Shin, M. Okada, S.
Fukumoto, M. Haraguchi, N. Takeda, K. Fujimura, M. Sakae, M. Kishikawa, H.
Shiku, K. Furukawa, S. Aizawa, Mice with disrupted GM2/GD2 synthase genelack complex gangliosides but exhibit only subtle defects in their nervous
system, Proc. Natl. Acad. Sci. USA 93 (1996) 10662–10667.
[10] M. Okada, M. Itoh Mi, M. Haraguchi, T. Okajima, M. Inoue, H. Oishi, Y. Matsuda,
T. Iwamoto, T. Kawano, S. Fukumoto, H. Miyazaki, K. Furukawa, S. Aizawa, K.
Furukawa, b-Series ganglioside deficiency exhibits no definite changes in theneurogenesis and the sensitivity to Fas-mediated apoptosis but impairs
regeneration of the lesioned hypoglossal nerve, J. Biol. Chem. 277 (2002)
1633–1636.
[11] T. Okuda, N. Tokuda, S. Numata, M. Ito, M. Ohta, K. Kawamura, J. Wiels, T.
Urano, O. Tajima, K. Furukawa, K. Furukawa, Targeted disruption of Gb3/CD77
synthase gene resulted in the complete deletion of globo-series
glycosphingolipids and loss of sensitivity to verotoxins, J. Biol. Chem. 281(2006) 10230–10235.
[12] Y. Kondo, N. Tokuda, X. Fan, T. Yamashita, K. Honke, H. Takematsu, A.
Togayachi, M. Ohta, Y. Kotzusumi, H. Narimatsu, O. Tajima, K. Furukawa, K.
Furukawa, Glycosphingolipids are not pivotal receptors for Subtilase cytotoxin
in vivo: sensitivity analysis with glycosylation-defective mutant mice,
Biochem. Biophys. Res. Commun. 378 (2009) 179–181.
[13] J. Boffey, D. Nicholl, E.R. Wagner, K. Townson, C. Goodyear, K. Furukawa, K.
Furukawa, J. Conner, H.J. Willison, Innate murine B cells produce anti-

Y. Kato et al. / Biochemical and Biophysical Research Communications 391 (2010) 750–755
disialosyl antibodies reactive with Campylobacter jejuni LPS and gangliosides
that are polyreactive and encoded by a restricted set of unmutated V genes, J.
Neuroimmunol. 152 (2004) 98–111.
[14] J. Boffey, M. Odaka, D. Nicoll, E.R. Wagner, K. Townson, T. Bowes, J. Conner, K.
Furukawa, H.J. Willison, Characterisation of the immunoglobulin variable
region gene usage encoding the murine anti-ganglioside antibody repertoire, J.
Neuroimmunol. 165 (2005) 92–103.
[15] T.R. Henion, D. Zhou, D.P. Wolfer, F.B. Jungalwala, T. Hennet, Cloning of amouse beta 1,3N-acetylglucosaminyltransferase GlcNAc(beta 1,3)Gal(beta1,4)Glc-ceramide synthase gene encoding the key regulator of lacto-series
glycolipid biosynthesis, J. Biol. Chem. 276 (2001) 30261–30269.
[16] C.J. Wikstrand, X.M. He, G.N. Fuller, S.H. Bigner, P. Fredman, L. Svennerholm,
D.D. Bigner, Occurrence of lacto series gangliosides 30 -isoLM1 and 30 ,60 -isoLD1
in human gliomas in vitro and in vivo, J. Neuropathol. Exp. Neurol. 50 (1991)
756–769.

755[17] C.J. Wikstrand, D.C. Longee, R.E. McLendon, G.N. Fuller, H.S. Friedman, P.
Fredman, L. Svennerholm, D.D. Bigner, Lactotetraose series ganglioside 30 ,60 isoLD1 in tumors of central nervous and other systems in vitro and in vivo,
Cancer Res. 53 (1993) 120–126.
[18] C.J. Wikstrand, P. Fredman, L. Svennerholm, D.D. Bigner, Detection of gliomaassociated gangliosides GM2, GD2, GD3, 30 -isoLM1 30 ,60 -isoLD1 in central
nervous system tumors in vitro and in vivo using epitope-defined monoclonal
antibodies, Prog. Brain Res. 101 (1994) 213–223.
[19] J.E. Månsson, P. Fredman, D.D. Bigner, K. Molin, B. Rosengren, H.S. Friedman, L.
Svennerholm, Characterization of new gangliosides of the lactotetraose series
in murine xenografts of a human glioma cell line, FEBS Lett. 201 (1986) 109–
113.
[20] Y. Kato, G. Jin, C.T. Kuan, R.E. McLendon, H. Yan, D.D. Bigner, A monoclonal
antibody IMab-1 specifically recognizes IDH1R132H, the most common gliomaderived mutation, Biochem. Biophys. Res. Commun. 390 (2009) 547–551.