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Enzyma-linked immunosorbent assay for antibody against the nicotinic acetylcholine receptor in human myasthenia gravis.

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Enzyme-Lmked Immunosorbent Assay
for Antibody Against the Nicotinic
Acetylcholine Receptor in Human
Myasthenia Gravis
Sachiko Kawanami, MD, Rieko Tsuji, BS, and Kenichiro Oda, M D t
Antibody against acetylcholine receptor (AChR) of human skeletal muscle was measured using enzyme-linked immunosorbent assay and found in 23 (74%) of 31 Japanese patients with generalized myasthenia gravis. In 15 patients
with generalized myasthenia gravis who had not undergone thymectomy and who were not receiving adrenocorticosteroids, the antibody was found in 13 (87%). Antibody was also found in 13 (54%) of 24 patients with myasthenia
gravis against AChR fractions obtained from fetal calf thymus. Based on the subunit structures of the AChR protein,
the double precipitation assay using iodine 125-a-bungarotoxin is also capable of detecting antibody against the toxin
binding site, by cross reactivity. This is among the first reports of experiments in which enzyme-linked immunosorbent
assay was used to measure the antibodies in human myasthenia gravis and provides evidence of anti-AChR antibody
against antigens from fetal calf thymus.
Kawanami S, Tsuji R, Oda K: Enzyme-linked immunosorbent assay for antibody against the nicotinic
acetylcholine receptor in human myasthenia gravis. Ann Neurol 15:195-200, 1984
Myasthenia gravis (MG) is an autoimmune disease
characterized by easy fatigability and weakness of
voluntary muscle resulting from defects in neuromuscular transmission. Antibodies against the nicotinic
acetylcholine receptors (AChRs) have been detected in
the sera of patients with M G [3, 19, 211, and passive
transfer of the disease from patients to animals by
humoral antibody [ 2 5 ) has been reported. The antibody was originally described as a serum factor that
blocks the binding of a-bungarotoxin (a-BuTx) to
acetylcholine (ACh) binding sites 12, 41. The presence
of the antibody has been assessed by radioimmunoassay using a complex of solubilized AChR and iodine
125-a-BuTx as antigen. This approach does not allow
for identification of antibodies directed against ACh
binding sites or the a-BuTx binding site [16, 22). The
antibody titers measured by this technique-that is,
the double precipitation method-do
not correlate
well with clinical severity 119).
Attempts have been made to detect antibodies reacting with the ligand binding site by measuring the inhibition of ‘251-labeled a-BuTx fixation of the purified
AChR, using sera from myasthenic patients 18, 12, 191.
These studies indicate a lower positive ratio of the
blocking antibody than of antibody directed against
sites other than the ACh binding site in the sera of
patients with MG. The antibodies to the homologous
AChR were considered to be directed predominantly
at sites on the AChR other than the ACh binding
site. In experimental autoimmune myasthenia gravis
(EAMG), Zurn and Fulpius [29} suggested, the subpopulation of antibodies directed against the toxin
binding site of the receptor may play a role in the
occurrence of paralysis after immunization.
We attempted to look for antibodies directed against
the toxin binding site of the receptor using enzymelinked immunosorbent assay (ELISA), without radioisotope or a-BuTx, in human MG. To our knowledge,
there has been one report in which ELISA was used
in EAMG [ 2 3 ] .
From the First Department of Internal Medicine, School of
Medicine, Fukuoka University, 45-1,7 chome Nanakuma, Jonan-ku,
Fukuoka 814-01, and the *Department of Internal Medicine, Saga
Medical College, Saga 840-01, Japan.
Received Nov 16, 1982, and in revised form Dec I , 1982, and May
17, 1983. Accepted for publication July 17, 1983.
Materials and Methods
ELISA
The method used for the anti-AChR antibody assay was a
“sandwich” assay reported by Engvall and Perlmann [9, 101.
ELISAs reported by hie and colleagues [13] for antibody
against myelin basic protein and for hepatitis B antigen by
Walters and co-workers [28} were used after some
modification. The wells of flat-bottom microtiter plates
(Cooke) were filled with 100 +I of AChR fraction with a
Address reprint requests
tO
D~ Kawanami.
195
protein concentration of 1 pghnl. The fluid was removed
from the plates after overnight incubation at 4"C, and the
plates were washed four times with phosphate-buffered
saline (PBS) containing 0.05% (vlv) Tween 20 (buffer A).
Serial dilutions of test serum were made in PBS containing
2% (wiv) bovine serum albumin and 0.05% (viv) Tween 20
(buffer B). One hundred microliters of test serum was added
to each well, and the preparation was incubated for two hours
at 37°C. Wells treated with buffer B alone were included in
the assay to determine the background. After dumping and
washing, 100 pI of the peroxidase conjugated antihuman immunoglobulin G rabbit serum (Miles Laboratories), diluted
1: 600 with buffer B, was added, and incubation was allowed
to proceed for a further two hours at 37°C. Excess conjugate
was washed out with buffer A, and 300 p1 of enzyme substrate solution was added to each well. The substrate solution
for peroxidase assay was prepared by dissolving 40 mg of ophenylene-diamine in 1 ml of methanol and then adding this
solution to 99 ml of distilled water and 30 pI of 309J hydrogen peroxide. The preparation was mixed in the dark and
adjusted to a p H of 6.0 with 1N sodium hydroxide. After the
preparation was incubated for one hour at a room temperature of 2O"C, the reaction was stopped by adding 25 ~1 of 8N
sulfuric acid. The yellow-orange color of the product of the
enzyme was read by the eye, and its extinction at 490 nm was
measured with a Mini Reader (Dynatech Lab. Inc). After
measuring the extinction, the ratio E was calculated as:
E =
E test serum - E blank I1
E blank I - E blank I1
where E blank I is the average extinction of five normal
negative control sera and E blank 11 is the average extinction
of the 100 p1 of the solution containing o-phenylenediamine, hydrogen peroxide, and sulfuric acid, as described.
When E was above 2.5, it was considered to represent a
positive reaction. The antibody titer in the ELISA was assessed by the serial dilution number of the sera that gave a
positive reaction.
Subjects
Serum samples were obtained from 37 Japanese patients with
MG, 27 women and 10 men. Among them, 31 patients were
grouped into clinical classes of Osserman type IIB and 6 cases
were of the ocular type. Four serum specimens were obtained during myasthenic crisis. All patients with generalized
MG were on a regimen of cholinesterase inhibitors. Thirteen
patients were receiving prednisolone, every other day. Eight
had undergone thymectomy, and 5 had thymoma. Fourteen
healthy subjects were tested as controls.
AChR Protein
The AChR fraction was obtained from fetal calf thymus or
human skeletal muscle using the method described by N e t t
and colleagues [ 151 for Elei-trophorus electricus, with two
modifications: ( 1) Brij 35 at a concentration of I:% (wiv) was
used as the solubilizing agent; (2) the diethylaminoethyl cellulose chromatography step was omitted. The human skeletal
muscle was obtained following radical mastectomy or leg
amputations. The specific activity of AChR protein was
196 Annals of Neurology Vol 15 No 2 February 1984
A
A
AA
A
Fig 1. Anti-acetylcholine receptor antibody measured by enzymelinked zmmunosorbent assay. Acetylcholine receptorfractions nbtained from fetal calf thymus were used as antigen. (MG = mya h e n i a gravis; D.N. = dilution number; TA( + ) = patients
witb thymoma; TA( - ) = patientJ. without thynioma.)
measured by a-BuTx binding assay [l}.The activities of fetal
calf thymus and skeletal muscle were 0.4 to 0.8 X 10 mol
per gram of protein and 320 x 10 mol per gram of protein, respectively (S. Kawanami and T. Horio, unpublished
data, 1982).
Disc gel electrophoresis of the fraction from the hydroxylapatite chromatography was performed to assess the
purity of the receptor preparations. The AChR protein fraction obtained by hydroxylapatite chromatography was dialyzed to 62.5 mM Tris hydrochloric acid, p H 6.8, containing
0.1% sodium cholate, then put on the disc electrophoresis in
5% polyacrylamide gel, using the method of Davis [6j.
Separating and stacking gels and the electrode buffer contained 0.1% sodium cholate. The electrophoresis was conducted at 4°C. The gel was stained with 1% amido black.
-'
Results
Anti-AChR antibody was detected with ELISA, using
AChR fractions from fetal calf thymus as antigen (Fig
1). Antibody titer was expressed by the dilution nurnber of the test serum that had a positive enzyme reaction, as described in t h e Materials and Methods SE'Ction. O n e of t h e 14 normal control subjects, a man w h o
is apparently healthy with n o signs or symptoms of
MG, showed a positive result. In t h e 6 patients with
ocular MG, there was n o evidence of a positive antibody. Of 24 patients with generalized M G , 13 pos-
28-
t
\
MUSCLE
( HUNAN)
Fig 2. Anti-acetylcholine receptor antibody measured by enzymelinked immunosorbent assay; comparison of titers using antigens
obtainedfrom fetal caFthymus and human skeletal muscle. The
same sera were tested with the two antigens. D . N . = dilution
number; Ag = antigen.)
sessed the antibody (54%). Patients with thymoma
showed a higher titer and frequency of the antibody.
Because antigen from fetal calf thymus is different in
species from that of humans and the specific activity is
lower than that of muscle, we used AChR fractions
from human skeletal muscle as the antigen. The same
samples were used to measure the anti-AChR antibody
with ELISA against rhe AChR fractions obtained both
from fetal calf thymus and human skeletal muscle (Fig
2). The antibody titer was higher in 5 of 8 cases when
the antigen from human muscle was used.
The results of disc electrophoresis of the receptor
preparations are demonstrated in Figure 3. AChR fractions from fetal calf thymus and human skeletal muscle
appeared in a single band.
When AChR fractions from human muscle were
used as the antigen for ELISA, a higher percentage of
the patients with M G showed the antibody (Fig 4).The
1 healthy control subject showing a positive result was
the same man in whom the antibody was detected using the antigen from fetal calf thymus. In generalized
MG, 23 of 31 patients (74%) showed positive reactions. A higher proportion of positive reactions-13 of
15 (87%)-was found in the patients with generalized
MG who had not undergone thymectomy and who
Fig 3. Polyactylamide disc gel electrophoresis of the isolated ucetyicholine receptor (AChR)fraction by ajjinity chromatographji
with cobrotoxin-Sepbarosefrom buman skeletal muscle and fetul
&thymus. Discs 1 and> show the results of mrkerproteins.
Discs 2 and 3: A C h R fraction from human skeletui muscle applied 40 pg in 100 ,uland 80 pg in 200 d,
respectioehl. Disc
4: AChR fraction from fetal calf thymus applied 180 p g in 200
pl. A indicates origin; E, dye top; B and C, bands of apoferritin
in discs 1 and 5 ; D, bands of botine serum aibumin in discs 1
and 5 .
were not receiving adrenocorticosteroids, however.
None of these patients had a thymoma. Four patients
showed the highest titer during myasthenic crisis. In
the patients with generalized M G and thymoma, there
was a tendency toward higher titers, as was also the case
when AChR fractions of fetal calf thymus were used.
In 4 patients with ocular MG, no positive reactions
were found.
The antibody titer decreased after steroid therapy. In
the patients not receiving steroids, a positive ratio was
seen in 19 of 21 (70%). In 5 patients anti-AChR antibodies were studied before and after prednisolone
therapy (Fig 5). There was no clinical improvement
when the antibody titer decreased, but three to six
months later there was a clinical recovery. When the
anti-AChR antibody was present, the titer increased in
parallel with the I g G concentration in the sera (Fig 6).
In the same sera the antibody titer against AChR
Kawanami et al: ELISA for AChR Antibody in MG
197
1.14.
2b
--
D.N.
26 .
0
0
27 -25 .
0
26 --
z4
-- 0
23 .
z4 --
--
22
--
2
0
22 .
00
23
0
I
0
0
--
,
1000
0
0
1
2000
300(
1gG
0
DL
Fig 6. Correlation between the titer of anti-acetylcholine receptor
antibody as measured by enzyme-linked immunosorbent assay
(ELISA)and serum immunoglobulin G. The serum IgG was detected by immunoprecipitation. Acetylcholine receptor protein from
human skeletal muscle was used as the antigen i n the ELISA.
O n
(-1
-80.1
NOQMAL
CONTRC
Fig 4. Anti-acetylcholine receptor antibody in human myasthenia gravis measured by enzyme-linked immunosorbrnt assay.
Anti-acetykcho/rne receptor antibod31 was measured as described
in the Materials and Methods section, using the acetylcholine receptor fractions from human skeletal muscle as the antigen. Open
circles indicate patients with myasthenia gravis and thymoma;
an asterisk under a circle indicates that the serum was obtained
during myasthenic crisiJ. (MG = myasthenia gravis; D.N. =
dilution number of serum; TX = thymectomy; S.T. = steroid
therapy, involving administration of prednisolone.20 to 60 mg,
evey other day.)
221
D.N.
27 .
26
.
25
.
24
.
23
-
5
10
15
PROLIML
Fig 7 . Correlation between anti-acetylcholine receptor (AChR)
antibody titers as measured by enzyme-linked immunosorbentarsay (ELISA) and by the double precipitation method. Titerr of
anti-AChR antibody detected by the double precipitation method
using AChR solubilized from human skeletal muscle as antigem
are shown along the abscissa. The ordinate indicates the antibody
titers demonstrated by ELISA using the same antigen.
22 .
2
.
~
(
G
)
1976
1
'77
I
'78
I
'79
1
'80
I
'81
. .
&I
'82
Fig 5 . Effects of steroids on anti-acetylcholine receptor antibody
measured by enzyme-linked immunosorbent assay. Sera were
stockedfrozen at -20°C. pike patients with myasthenia gravis
were studied before and after administration of prednisolone,20
to 60 mg, evey other day. Dashed lines show findings after initiation of steroid therapy. (TA( + ) = myasthenicpatient with
thymoma who underwent thymectomy in 1979; TA( - = patients without thymom.)
198 Annals of Neurology
Vol 15 No 2 February 1984
from human skeletal muscle determined by ELISA was
compared with the value of antibody detected by double precipitation assay using complexes of '251-a-BuTx
and the same AChR fraction (Fig 7). The sera with
lower antibody titers as determined by the double precipitation method showed higher titers on ELISA.
Discussion
Antibodies against AChR, including the toxin binding
site, were detected by ELISA. When AChR fractions
from human skeletal muscle were used as antigen, 1')
(90%) of the 2 1 patients with generalized M G showed
positive results, these positive findings being absent in
the sera of 6 patients with ocular myasthenia. The patients with thymoma had higher titers of the antibody.
These results are similar to findings in previous studies
performed with radioimmunoassay using a complex of
1 2 5 1 - a - B ~ Tand
~ AChR as antigen. A higher positive
ratio in the myasthenic patients was found with ELISA,
as compared with the inhibitory assay of fixation of 1251labeled a-BuTx to purified AChR.
The radioimmunoassay double precipitation technique is thought to be capable of detecting only antibodies against AChR, other than toxin binding sites
{19, 22). AChR purified from the electric organ of
Torpedo californzca is composed of four subunits in the
mole ratio a&y6 {IS, 271. These subunits are acidic
glycopeptides and have apparent molecular weights of
38,000, 50,000, 57,000, and 64,000 for 01, 6, y , and 6,
respectively. The 01 subunit contains the ACh binding
site in the electric organ of Torpedo or Electrophorzls and
also in skeletal muscle AChR [ S , 11, 17, 18, 20, 241.
One or more other subunits may be involved in the ion
channel, the opening of which is regulated by ACh
binding {5). Using monoclonal antibodies against Torpedo californica and its subunit, Tzartos and Lindstrom
1261 noted a similarity between a and p subunits and
between y and 6 subunits. Antisera to these subunits
with a similar molecular weight showed cross reactivities among various species. These findings suggest
that the antibody to the ACh binding site-that is,
anti-ACh site antibody-can bind to the complexes of
1 2 5 1 - a - B ~ Tand
~ AChR at the portion of p subunits,
after the antibody binding sites on a subunits have
been occupied by ' 2 5 1 - ~ - B ~ TTherefore,
~.
when the
double precipitation assay is used, the anti-ACh site
antibody may be measured simultaneously with antibodies other than those to the ACh binding site. It
appears that the antireceptor antibodies are directed at
the ACh binding site of the receptors, as determined
by this method. The positive ratio of the antibody in
generalized M G determined by ELISA and the double
precipitation method was similar.
Antibodies detected by inhibition of toxin binding
were found in only 38% of the patients with M G [19].
With this method, AChR extract was incubated overnight with test serum globulin, '251-labeled a-BuTx was
added, '251-a-BuTx-AChR was separated, and the
radioactivity was measured. The binding of 1251-aBuTx may be inhibited after formation of an immune
complex by the anti-AChR antibody against its p subunit.
ELISA permits detection of antibodies against whole
subunits, including the ACh binding site, and seems
more pertinent and sensitive than the inhibitory assay
using radiolabeled 01-BuTx or double precipitation assay.
The highest titer was seen during myasthenic crisis,
whereas in patients receiving prednisolone, there was a
longer interval before clinical improvement after elimination of the antibody from the serum. The antibody
against AChR may relate only partly to the neuromuscular transmission block at the chronic stage of the
disease. The formation of immune complexes may destroy the receptor but does not seem to disturb
neuromuscular transmission when the complex is produced.
We found anti-AChR antibody against antigens obtained from fetal calf thymus. The origin of the antiAChR antibodies in M G remains unknown. These results suggest that the thymus gland may be the site of
the primary lesion in M G and that lymphocyte functions may be altered in patients with this disease {7,
14).
The authors thank Drs Y .Nagai and H. lrie for helpful advice, D r K.
Hayashi for the gift of cobrotoxin, Drs Y . Kuroiwa, R. Mori, M.
Okumura, and K. Nishimaru for support, and M. Ohara for reading
the manuscript.
Supported by Grant 557206 from the Japanese Ministry of Education and by a grant from the Cancer Foundation of Fukuoka Prefecture.
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