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Serum IgG Antibody to
Ganglioside GQIb Is a
Possible Marker of Miller
Fisher Syndrome
Atsuro Chiba, MD, Susumu Kusunoki, MD,
Teruo Shirnizu, MD, and Ichiro Kanazawa, M D
We studied serum anti-glycolipid antibodies by enzyme-
linked immunosorbent assay and thin-layer chro-
matography-enzyme immunoassay in six consecutive
patients with typical Miller Fisher syndrome. In all six,
increased activity of IgG antibody against ganglioside
GQ1b was present i n the early phase and reduced with
time, whereas such activity was not detected i n normal
control subjects and disease control subjects including
those with GuiIlain-Barre syndrome. Anti-GQ l b IgG
antibody is a new possible diagnostic marker of Miller
Fisher syndrome and could well be related to the disease
process itself.
Chiba A, Kusunoki S, Shimizu T, Kanazawa I.
Serum IgG antibody to ganglioside G Q l b is a
possible marker of Miller Fisher syndrome.
Ann Neurol 1992;31:677-679
Miller Fisher {l] reported three patients with a syn-
d r o m e of ophthalmoplegia, ataxia, and areflexia, and
From the Department of Neurology, Institute of Brain Research,
School of Medicine, University of Tokyo, Tokyo, Japan.
Received Sep 3, 1991, and in revised form Nov 18 and Dec 27.
Accepted for publication Dec 30, 199 1.
Address correspondence to Dr Kusunoki, Department of Neurol-
ogy, Institute of Brain Research, School of Medicine, University of
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113, Japan.
Copyright 0 1992 by the American Neurological Association
Guillin-BarrC syndrome (GBS). Although the precise
relationship between these two syndromes is un-
known, they have t h e following c o m m o n clinical fea-
tures: frequent Preceding infectious episode, a benign
clinical course with -good recovery, and Dossible im-
m u n e mediation. Recently anti-glycolipid antibodies
were reported in patients with GBS {2-61. We studied
anti-glycolipid antibodies in patients with Miller Fisher
syndrome tO investigate their relationship from this
immunological viewpoint.
Materials and Methods
We studied six patients with typical Miller Fisher syndrome.
Their clinical features are summarized in the Table. They all
had a preceding infectious episode, ophthalmoplegia, ataxia,
hypo- or areflexia, and a benign clinical course with good
recovery. Patients 5 and 6 showed mild weakness of proximal
upper extremities that rapidly recovered. No patient showed
abnormalities on nerve conduction studies. Sera were ob-
tained serially as indicated in the Table. Sera were also taken
from 16 normal control subjects, 16 patients with GBS, 14
patients with multiple sclerosis (MS), and 14 patients with
other immunological disorders (OID) including nine with
systemic lupus erythematosus and five with polyrnyositis. All
sera from disease control subjects were taken in the acute or
active phase. One patient with GBS had a right abducens
palsy, but no patient with GBS demonstrated ataxia.
Enzyme-linked Immunosorbent Assay I E L I S A )
Ten different purified glycolipids (GM1, GM2, GM3, G D l a ,
G D l b , GD3, G T l b , G Q l b , asialo-GM1 [GAl), galactoce-
rebroside) were used as test antigens. Glycolipids except for
GA1 were purchased from Funakoshi (Tokyo, Japan). GA1
was prepared from GM1 of bovine brain in our laboratory.
Five hundred nanograms of purified ganglioside in 50 p,1 of
ethanol was added to each well in Linbro microtiter plates
(Flow Laboratories, McLean, VA), and the solution was dried
by evaporation. Nonspecific protein binding sites were satu-
rated with 1% bovine serum albumin in phosphate-buffered
saline (PBS), p H 7.4 (blocking solution). Fifty microliters of
the sera diluted 1:40 with blocking solution was added in
duplicate to each antigen-coated well. The plates were incu-
bated overnight at 4°C. Then the wells were washed three
times with the blocking solution and incubated with 50 I J . ~of
peroxidase-conjugated goat anti-human IgG or IgM (Cappel,
West Chester, PA) diluted 1 : 500 or 1 : 200, respectively,
with the blocking solution for 1 hour at room temperature.
Then the wells were washed three times and incubated
with 40 mg/dl of o-phenylenediamine dihydrochloride and
0.006% H202in phosphate-citrate buffer, p H 5.0, as an
enzyme substrate. The reaction was stopped by 50 ~1 of 8
N H,SOj and the color reaction was read at 492 nm using
an ELISA reader (BioRad, Hercules, CA). Antibody activity
was expressed as the mean optical density (OD) of the dupli-
cate wells. The blocking solution, instead of diluted sera, was
incubated in uncoated wells followed by the same procedure,
and the obtained OD was used as a blank.
677
Clinical Features of the Patients with Miller Fither Syndrome
Patient No. 1 2 3 4 5 6
Ageisex 621F 5GIM 6GlF 431M 411M Si5lM
Type o f preceding infection R R R R R I<
Ophthalmoplegia + + + + + -t
Hyporeflexia or areflexia + + + + + -t
Ataxia + + + + + -t
Recovery CR CR SR CR SR SR
Serum sampling
First’ 31121 1612 813 1619 37133 1212
Follow-upc 17 11 14 8 13 11

’Corresponding to Figure 2.
bDays from the onset of the preceding infecrionineurological symptoms
‘Days after the first sampling.
R = respiratory symptoms; CR = complete recovery; SR = still recovering

Thin-Layer Chroniatograpby (TLC) and

Enzyme lrnmunoassay
Each 1 ~g of purified G D l b and G Q l b was loaded on a
plastic-backed TLC plate (Macherey-Nagel, Doren, Ger-
many), and then the plate was developed with chloroform1
m e t h a n o l i 0 . 2 ~CaC1, (45 : 55 : 10). The plate was air-dried,
dipped in a solution of 0.4% polyisobutylmethacrylate in
n-hexane, and air-dried again. Different lanes on the plate
were overlaid with the patient’s or with control sera diluted
1 : 40 with the blocking solution and incubated overnight at
4°C. Then the plate was washed with PBS and incubated with
peroxidase-conjugated goat anti-human IgG antibody diluted
1 : 300 for 2 hours at room temperature. Immunoreactants
were visualized with 50 mgldl of 3,3’-diaminobenzidine tet-
Fig I . Antigenic spec;fcity in IgG class. The top designations
rahydrochloride and 0.01% H,Oz in PBS.
indicate antigens with which the well5 in each column are
coated. The solid line (-1 and GC mean blank tantigen-
Results uncoated) and galactocerebroside, respectively. The sera of pa-
Anti-GQlb IgG activity was found in all six patients tients with Miller Fisher syndrome (Patients l t o 6 ) all reczct
with Miller Fisher syndrome (Fig 1). In Patient 2 , anti- with GQlb. For Patient 2, anti-GDlb IgG activity is aho de-
G D l b IgG activity was also detected. The reactivities tected. Although the serum of the patient with Guillain-B‘zrri
syndrome (GBSj, who had a preceding histoy of Campylobacter
were confirmed by the TLC-enzyme immunoassay.
enteritis, reacts with G M I , neither the normal control subject
None showed IgG activity against the other glycolipids
nor the patient with GBS shows reactivity t o GQ 16.
or IgM activity against any glycolipid used in this study.
The anti-GQlb IgG activities by OD were all three
standard deviations greater than those of normal con-
trol subjects, whereas none of the disease control
groups’ were above normal (Fig 2). The mean OD of anti-GQlb IgG activity was detected in the early phase
the Miller Fisher syndrome group was significantly of all six patients with the syndrome, but not in normal
higher than those of the normal and disease control control subjects, patients with GBS, and other disease
groups ( t test, p < 0.0001). Serial studies showed that control subjects. The high incidence, the specific na-
the anti-GQIb IgG activities reduced with time in all ture in this syndrome, and the fall of activity with time
patients with Miller Fisher syndrome. all suggest an association between anti-GQIb IgG anti-
body and this disorder.
Discussion Several authors have investigated anti-ganglioside
This is the first report of anti-glycolipid antibodies in antibodies in patients with GBS E2-61. The incid’ence
patients with Miller Fisher syndrome. Increased serum of positives varies and the antigenic specificity deplends

678 Annals of Neurology Vol 31 No 6 June 1992


.;i A
10
Fig 2. Anti-GQl b IgG activity in patients with Miller Fisher
syndrome. in noma/ control subjects, in patients with
Guillain-Barre'syndrome, and in other disease control subjects.
The follow-up data of the patients with Miller Fisher syndrome
are also shown. The h y s on which serum was sampled in pa-
tients with Miller Fisher syndrome are mentioned in the Table.
The horizontal broken line indicates the mean + 3 SDs of the
normal control subjects. The ,figures in parentheses at the bottom
express the mean ? SD of each group. MS = multiple sclerosis;
OID = other immunological disorders.
on the patient. In our series, anti-glycolipid antibodies
were detected in about two-thirds of the patients with
GBS and GM1 was the most commonly recognized
antigen [b}. But anti-GQlb antibody has not been re-
ported in patients with GBS yet, and it was not de-
tected in this study. The clinical picture of GBS is not
homogeneous in its preceding infectious episode, the
combination of symptoms and signs, or histopathologi-
cal and electrophysiological findings. Recently, Yuki
and colleagues [ 5 ] reported anti-GM1 IgG antibody in
two patients with an acute axonal form of GBS after
Campylobacter enteritis. We have also seen two pa-
tients with GBS after Campylobacter enteritis, both
of whom had monospecific anti-GM1 IgG antibody
(Kusunoki S, unpublished data). This finding suggests
some association between antigenic specificities of
anti-glycolipid antibodies and clinical forms of GBS.
The relationship between Miller Fisher syndrome
and GBS is not fully understood. Our findings show
that Miller Fisher syndrome has an immunological fea-
ture in common with certain forms of GBS, namely
the presence of anti-ganglioside antibodies. However,
the antigenic specificities are different between the two
syndromes; anti-GQlb IgG antibody was detected spe-
cifically in Miller Fisher syndrome.
G Q l b is a relatively minor component of the gangli-
Brief Communication: Chiba et al: Anti-GQlb Antibody in Miller Fisher Syndrome 679
osides in both central and peripheral nervous systems.
In the human central nervous system, G Q l b is present
more in neurons than in the myelin 171. This tendency
is also suspecred in the peripheral nerves of rats {8].
However, the specific distribution of G Q l b is not yet
known.
Further studies are needed to understand fully the
significance of this anti-GQIb IgG antibody. Whether
it is neurotoxic or is otherwise directly responsible for
the neurological symptoms different from those of
GBS, or is an incidental by-product of an yet unde-
scribed immune process that is perhaps related to the
preceding infectious episode, all remains to be eluci-
dated. Regardless, the specific appearance of this anti-
G Q l b IgG antibody in Miller Fisher syndrome sug-
gests that this is a possible immunological marker of
the syndrome and could be of great clinical value.
This work was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, and Culture of
Japan (02259203, 02770445) and a grant from the Ministry of
Health and Welfare of Japan.
We thank D r Takeshi Hayakawa, Yoyogi Hospital, D r Tomoji Wata-
nabe, Toranomon Hospital, D r Humihiko Sato, Yanagihara Hos-
pital, Dr Syuji Nishimura, Tokyo Metropolitan Police Hospital, D r
Keiichiro Nakano, Department of Medicine and Physical Therapy,
School of Medicine, University of Tokyo, and D r Hideji Hashida,
Dr Keiko Ide, D r Yasuhisa Sakurai, D r Tadashi Komiya, Dr Tai
Miyazaki, and Dr Mitsuru Kawai of our department for giving us
the opportunity to investigate their patients' sera.
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