Vol. 88, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATlONS
May 28, 1979
Pages 575482
MONOCLONAL ANTIBODIES AGAINST PURIFIED NICOTINIC ACETYLCHOLINE RECEPTOR Christopher
M. Gomez',
David
Barry 1 Department
1* , Phillip
P. Richman
G.W. Arnasonl
W. Berman',
and Frank
2 Neurobiology Received
Laboratory, March
Salk
Institute,
A. Burres',
W. Fitch3
of Neurology and 3Department of Pathology, Sciences and the Pritzker School of Medicine, Chicago, Illinois 60637
Biological of Chicago,
Steven
San Diego,
Divisitm of the The University
California
92112
12,1979 SUMMARY
Eleven stable monoclonal hybridoma cell lines synthesizing antibodies against purified acetylcholine receptor from Torpedo californica were Spleen cells from a rat immunized with acetylcholine receptor produced. and exhibiting experimental autoimmune myasthenia gravis were fused with a mouse myeloma cell line: Studies of the binding of these antibodies to acetylcholine receptor by use of a passive hemagglutination assay in the presence of various cholinergic ligands revealed four general categories of binding specificities: 1) blockade only by alpha bungarotoxin, 2) partial blockade by all ligands, 3) increased titer .in the‘presence of alpha bungarotoxin and benzoquinonium chloride, 4) absence of effect by any ligand. INTRODUCTION Anti-sera useful
tools
zation
of these
of multiple
in
populations
a means by which be obtained. have
applied
ceptor (AChR). * To whom reprint
this
reagents
Recently
this
protein
should
developed
in this
of nicotinic has been
techniques
provided
and specificity manner
of macromolecular
to the study
to be
irmnunochemical
(2) have
structure
prepared
probes
in their
(mAbs)+
of uniform
to be powerful
requests
differ
antibodies
immunoglobulin
Although
which
have proven
and --in situ localigeneral, are made up
in
specificities.
of monoclonal
approach
antigens
Such anti-sera,
of antibodies
Immunochemical prove
purified
quantification,
(1).
and antigenic
the production
doubtedly
against
the identification, antigens
characteristics for
raised
will
may un-
structures. acetylcholine
intensively
studied
We reby
be addressed
t Abbreviations: mAb, monoclonal antibody: AChR, nicotinic acetylcholine receptor; EAMG, experimental autoimmune myasthenia gravis; aBT, alpha bungarotoxin; Cm, carbamylcholine; dtc, d-tubocurarine; atr, atropine sulfate; Bz, benzoquinonium chloride; Brij-PBS, phosphate buffered saline with 0.05% Brij 35; RBCs, sheeperythrocytes; HA, passive hemagglutination assay. 0006-291X/79/100575-08$01.00/0 575
Copyright All rights
@ 1979 by Academic Press, fnc. of reproduction in anyform reserved.
BIOCHEMICAL
Vol. 88, No. 2, 1979
biochemists
and biophysicists,
lationship
between
the subunit range
its
4 distinct
can be altered
through
gravis
develop
(EAMG),
Monoclonal ceptor
cule spleen
the model
cells a myeloma
lines
producing
of the binding
for
for
disease,
with
valuable
tools
identifying
regions agents,
the induction
from
rats
cell
of these
known to modulate
with
to produce
anti-receptor
ligands,
antibodies receptor
snake
neurotoxins, purified
autoimmune
Tor-
myasthenia antibodies
(6).
on the
surface
of re-
elucidating
the
subunit
structure
of the molecule defining
We report
to AChR inthepresence
for
on the mole-
described
AChR and exhibiting of stable
important sites
In the studies
a number
antibodies.
of which
with
of anti-AChR
to
areas
and for
of EAMG.
immunized
line
for
thought
the properties
experimental defined
Even
structures is
immunized
the appearance with
protein
(5)
animals
reactive
of pharmacologic
with
stances
In addition,
provide
crucial
states
the re-
unanswered.
proposed
This
the use of cholinergic
concomitant
protein
the binding
(3,4).
concerning
remain
unresolved,and
functional
antibodies
should
of this
of AChR is
anesthetics.
pedo AChR
questions
and function
1 and 4 polypeptides
have at least and local
fundamental
structure
composition
between
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
below,
EAMG were hybridoma
preliminary
fused
cell studies
of various
sub-
function. METHODS
Pharmacologic Agents - Alpha bungarotoxin (aBT), [lz51]aBT, and alpha neurotoxin were kindly supplied by Dr. James Patrick. Carbamylcholine (Cm), d-tubocurarine (dtc), atropine sulfate (atr), choline bromide and physostigmine were obtained from Sigma (St. Louis, MO), neostigmine methylsulfate from Elkins-Simms (Cherry Hill, NJ) and sodium acetate from Fisher (Fair Lawn, NJ). Benzoquinonium chloride (Bz) was a gift of Sterling Drug Company (Rensselaer, NY). All solutions were prepared daily. AChR Preparation - AChR was prepared from the electroplax of Torpedo californica by the method of Berman and Patrick(7,g). Approximately equal proportions of monomeric and dimeric AChR (3.8-4.0 nmoles of aBT binding sites per mg protein) were suspended in phosphate buffered saline containing 0.05% of the non-ionic detergent Brij 35 (Cal Biochem, San Diego, CA) (Brij-PBS). Immunization - AChR in 0.05% Brij-PBS was emulsified with an equal volume of complete Freund's adjuvant and injected in multiple intradermal sites along the back and in the footpads of 200 gm female Lewis rats. In the primary immunization each animal received 45ug of AChR protein. At the same time each animal was injected with forty-five microliters of killed B.pertussis vaccine (gift of Eli Lilly Co., Indianapolis, IN) at multiple intradermal sites. All animals developed the typical clinical course of EAMG(9) with frank weakness beginning on day 9-12 after inoculation and clearing by day 21. On the 24th day, at which time the acute phase of the disease had cleared but prior to development of the chronic phase, animals received a second injection of AChR, 25 to 1OOug without adjuvant, intraperitoneally. Spleen Cell Hybridization (10) - Three days after the second injection of AChR, spleens were removed from immunized animals, homogenized in a ground glass homogenizer, and centrifuged over a Ficoll-Hypaque gradient (S.G.: 1.094). The mononuclear cell suspension thus obtained was then fused with
576
Vol. 88, No. 2, 1979
BIOCHEMICAL
AND BfOPHYSICAL RESEARCH COMMUNICATIONS
the mouse myeloma cell line P3-X63-Ag8 (kindly provided by Dr. C. Milstein). Spleen cells (5 x 107) and myeloma cells (5 x 106) were mixed in 6Omm Petri dishes (Falcon) and centrifuged at 250xg for 3 minutes. The cells were then flooded with 50% polyethylene glycol (PEG 1500, Fisher, Fair Lawn, NJ), cells were cultured overnight in Dulbecco's modified and after washing, Eagles medium with 20% heat-inactivated fetal calf serum. The cells were then cultured in hypoxanthine-aminopterin-thymidine (HAT) medium in microtest wells (Costar, Cambridge, MA) (approximately 1 x lo4 cells per well). Supernatants from wells with proliferating hybrids were assayed (see below) for anti-AChR activity. Cells from positive wells were then cloned by limiting dilution in microtest wells with irradiated Lewis rat thymocytes serving as a feeder layer. Clones were grown up in bulk in spinner flasks. Some clones were re-cloned by limiting dilution. Antibody Assay - A passive hemagglutination assay (HA) was performed by modification of the method of Aarli et al. (11). AChR (T californica) in 0.05% Brij-PBS was coated onto sheep erythrocytes (RBCs) $eviously treated with 83.3ugfml tannic acid. The cells were then washed and resuspended in 0.05% Brij-PBS. The ligand-binding properties of the RBC-bound AChR were tested in a competitive binding assay. AChR-coated RBCs were incubated with various ligands (10B5M) in 0.05% Brij-PBS for 30 minutes prior to a 30-minute incubation with [1251]aBT (10e7M). The cells were centrifuged through a layer of n-butyl phthalate and the pellets counted in a gamma counter. Serum or culture supernatants were serially diluted with 0.05% Brij-PBS and 1% non-immune heat-inactivated rabbit serum. The AChR-coated sheep erythrocytes were then added to the serum (or supernatant) dilutions in round bottom microwell plates (Falcon, Oxnard, CA), incubated at 3J°C for 15 min., and scored for agglutination visually and also after centrifugation at 250xg for 1 min. (12). Assessments of blockade of antibody binding in the HA were carried out by preincubation of AChR-coated cells with pharmacologic a ents in 0.05% Brij-PBS or with 0.05% Brij-PBS alone B to their addition to the serum or supernatant for 30 minutes at 20 C prior dilutions. RESULTS Hybridization:
All
of spleen 217 .
cells
Half
from
were
layer
wells
grew within number that
that
(Bk) whose
serum HA titer
out hybrids.
Seventy
for
the well
as a single
antibody-producing
(see below) more than
wells dilution
were
with
from which from
the
they
were
same well
are
were
positive were
re-cloned for
antibody
monoclonal.
of the mAbs remained
lo%-30%
Clones
stable
to AChR was
percent
anti-AChR
hybridomas. patch.
clones
clones
contained
proliferating
well
derived (n=25)
the hybridization
rat
by limiting
the original
from
grew
hybrid-positive
of the primary
resulted
a single
positive
subclones
here
planted
obtained
some clones
these All
from
the wells
supernatants Clones
mAbs studied
of planted
All were
of the
activity. feeder-
such hybridomas
designated
derived.
by the
(It
is
possible
identical.)
Five
by limiting
dilution.
production
The binding
of
suggesting specificities
as the hybrids
were
carried
through
10 passages.
mAb Specificities:
The HA titers
of culture
receptor
are
in Table
hybridomas
presented
577
supernatants 1.
Most
from
supernatants
11 antiwere
Vol. 88, No. 2, 1979
8lOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1 Anti-AChR BA Titers (Log2) of Various Cholinergic Ligands * Ligand BZ aBT dtc GUI
Supernatant in the Presence Clone Control
atr
60D
13
0
13
12
12
13
561 56K 57L 625
29 22 16 23
24 20 14 18
24 21 14 18
21 22 14 20
24 20 14 20
21 21 14 18
73G
17
20
17
21
17
17
381 58E 58F 77A 77F
13 9 6 19 20
13 9 6 19 19
13 9 6 19 20
13 9 6 19 20
13 10 6 20 20
14 9 6 19 20
i
dtc,
assayed
Bz, Cm, atr - 10e514; aBT - 10m7M
at least
in which
3 times,
5 replicates
95% confidence clones
obtained
from
are not titers
RBCs in Brij-PBS
supernatants.
representative
carried
of these
cultures
Control coated
were
limits
between
with
out
titers
significant
since different
alone
Each supernatant
Differences
the supernatants cell
to the addition
was also
tested
the
in HA titers tested
were
densities.
of preincubation
of the AChRof antibody-containing
against
uncoated
RBCs was
detected.
The pharmacologic activity of RX-bound AChR was tested by radioassay (see Methods). Binding of ['251]aBT to AChR-
RBCs was inhibited
of effectiveness:
no agglutinating
activity
RBCs treated
coated
Brij-PBS;
Experiments
and tanned a competitive
with
the result prior
given.
on the same day determined to be +2l.
containing are
results
by cholinergic
ligands
in the
following
order
aBT>dtc>Bz>Cm>atr.
The effects of various cholinergic ligands on antibody binding in the HA are indicated in Table 1. In control experiments each ligand was tested for, and found to lack, agglutinating activity against the AChR-coated acetate,
RBCs. choline
Preincubation of the receptor-coated bromide, neostigmine methylsulfate,
578
RBCs with
sodium
and physostigmine
Vol. 88, No. 2, 1979
(all
BIOCHEMICAL
AND EIOPHYSICAL RESEARCH COMMUNICATIONS
10V5M)
had no effect on the HA titers of any of the hybridoma superThe data allow roughly four categories of mAb specificities In the first category, represented by hybridoma 60D, to be distinguished.
natants.
hemagglutination
was completely
ligands.
The second
57L,
was that
625)
and by all
other
antagonist,
type
creased
it.
from with
unaffected
The inhibitory
effect
60D was studied
half
The possibility following
that
from
reacted
Resuspension natant
with
the possibility against
that
30 minutes
natant
with
resulted
Free
aBT thus
cating
that
between
with
and addition
with
from
does not
affect
the antibody
AChR-coated
a competitive
equal is
for
of 625 superexperiment
actually
be directed
RBCs were
preincubated
inhibitor values
hemagglutination
10e7M c&I
agglutination.
of aBT binding.
30 minutes)
to control
directed
with
dilution
60D could
degrades
out by the
In another
clone
1)
6 x 10WvM and 8 x lo-'M.
no resultant
of a 1:27
aBT (10V7M
in HA titer
(Figure
preincubated
hemagglutination.
10V4M dtc,
incubation
the concen-
curve
of the RBCs was ruled
aBT and not AChR was tested.
Subsequent
by antibody
Increasing
60D supernatant
antibody
and Bz in-
tested.
inhibition
RBCs were
in definite
in which
381, 58E and F, and 77A and F
ligands
AChR-coated
of the cells
resulted
that
of aBT and 60D supernatant
the surface
by aBT
the muscarinic
however,aBT
detail.
the combination
it
blocked
of CYBT on hemagglutination a steep
by other
561 and K,
73G) is
hybridomas the
at aBT concentration
experiment.
and then
includes
in greater
effect
including
(hybridoma
by all
effect
(hybridomas
the HA titer;
the HA produced
maximal
AChR or releases
for
category
were
of aBT in
tested,
reduced
little
was partially
specificity
tested
The final
clone
tration
ligands
The third
HA titers
observed
hemagglutination
cholinergic
atr.
by aBT with
of specificity
in which
none of the ligands where
blocked
and then for
60D super-
60D (i.e.,
213).
by 60D supernatant,
against
AChR and not
indi-
against
the
toxin.
DISCUSSION The experiments tion
of the binding
purified bodies
nicotinic were
AChR could binding bodies dtc
which
in
several
cholinergic
of monoclonal
receptor.
studied,
and the binding
of six
by cholinergic
ligands.
could
completely competitively ways. ligands
inhibit tested
Four
aBT.
all
is bind
but
In the first
This that to the
579
not result
against
by other
of mAb type
of
RBCs by antiligands
such as
may be accounted
antibody same site
anti-
to erythrocyte-bound categories
of receptor-coated
by aBT,
One possibility
investigaprepared
hybridoma-derived
of these
be distinguished.
60D) agglutination inhibited
preliminary antibodies
Eleven
be altered (hybridoma
is
above represent
(13)
acetylcholine
specificities
specificity
presented
properties
60D, uBT, on AChR.
for
and the QBT is
BIOCHEMICAL
Vol. 88, No. 2, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2’0 2e-
? l-
26-
24-
a Bungarotoxin
Figure
to bind
to AChR in an essentially
other
ligands
studied
much less be similar
than
then
60D antibody
account
is
site.
from
possibility the
ligand
binding, the
site, would
binding
results
60D binding
site
73G are difficult sites
that
than
antibody explanation
in close
and would
proximity
be expected
Steric
hindrance
at locations would
60D binds
oBT binding,
60D which
any of the other
ligands
change
by
and 10e7M cd3T does
binding
antibody
would
hemagglutination
smaller
they
facilitation
towards are
Further
by ligands by antibodies
to interpret.
directed
or that
conformation.
predicted
antibody
and that
inhibition
of hemagglutination
either
Cm,
dtc,
ligands to
by
peripheral
not be effective.
at a site but not
in AChR which
remote
other
ligand
occludes
the
site.
and 625 and the apparent are
be that
in a conformational
The partial tested)
while
with
dtc
of the AChR surface.
to inhibit
binding
60D binds
60D binds
8,000)
the
than
and suggests An alternative
antibody
approximately
portion
be expected
that
while
antibody
that
aBT is much larger
after
affinity behavior
of lo-%
for
mask a considerable
A third
binding
in the presence
weight
If
The observation
the data
fashion,
much greater
interpretation binding site.
(molecular
aBT might
reversible. with
support this to the ligand binding
to the ligand
irreversible
of antibody
observed.
can occur
to the ligand
readily caBT but
the type
to that
not, however, does not bind
tested
are
affinity
and atr,
could
(Ml
1 - Anti-AChR HA titers of hybridoma clone 60D supernatant AChR-coated FzBC preincubation with various concentrations of CXBT
known
Bz,
Concentration
(at
the single
from hybridomas of hemagglutination
concentration 561 and K, 57L by hybridoma
These results suggest that the antibodies antigenic determinants close to ligand binding
capable
of detecting
investigations
studying
580
ligand-induced a wide
changes range
of ligand
in AChR con-
Vol. 88, No. 2, 1979
centrations, assay
and making
(7,8)
will
A somewhat tion
antagonist
observation
We know of no prior binds
to Torpedo
to compete
with
remote
with
very
differences
from high
characteristics blocking
assays than
identified
investigations
using
of cholinergic
from
tested.
sites,
or,
it
derive with
(15).
may bind they
from
may
structural
active
respect
(Preliminary
381,
Some of the
are particularly
differently
re-
AChR (14).
These
site.
is
hybridomas
conversely,
binding
in
to ligand
results
have
and 77F as IgM.)
of the monoclonal
antibodies
the nicotinic reagents
of AChR, ofthe
muscarinic
to Electrophorus
antibodies
IgM antibodies behave
of hemagglutina-
this
concentration
in the HA may also
as IgG-2a
these
that
ligands
ligand
possibilities.
blockade
binding
immunoglobulins
by which
these
at high
of the
binding
and might
60D and 625
and pharmacology
for
observed
do other
new tools
suggesting
to the
The availability provides
the partial
by the
the ligand affinity
of the mAbs.
agglutination
is
titers
such as radioimmuno-
between
AChR, however,
unaffected
in behavior
assays
data
a-neurotoxin
P, and 77A and F are to sites
antibody
to distinquish
The hemagglutination
bind
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
use of other
be required
surprising
by atr.
ported
8lOCHEMlCAL
described
AChR may be explored.
will
include
pathogenesis
studies
of the
above Future structure
of EAMG, and of the mechanisms
transmission. ACKNOWLEDGMENTS
We thank stages This
of this work
4197)
Dr.
project
was supported
and the Muscular
post-doctoral Fellowships
Fellowship from
Tom McKearn
for
his
and Susan Tonsor in part Dystrophy
extensive for
by grants the NIH
the Myasthenia
Gravis
excellent from
Association
from
help
technical
the NIH (NS13526,
of America,
(NSOS715) Foundation,
in the early
as well
and H.R. Viets
assistance. A19268, as a Student
Inc.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Yalow, R.S. (1978) Science =,1236-1245. Kohler, G. and Milstein, C. (1975) Nature =,495-497. Heidmann, T. and,Changeaux, J.P. (1978) Ann. Rev. Biochem, 67,317-357. Raftery, M.A., Vandlen, R.L., Reed, K.L., and Lee, T. (1976) Cold Spring Harbor Symp. Quant. Biol. x,193-202. Steinbach, J.H. (1979) Cell Surface Reviews, Poste, G., Nicolson, G. Publishing, Amsterdam, in press. and Cotman, C. eds., North Holland Patrick, J. and Lindstrom, J. (1973) Science =,871-872. Patrick, J.,Lindstrom, J., Culp, B., and McMillan, J.'(1973) Proc. Nat. Acad. Sci. USA z,3334-3338. J. Submitted for publication Berman, P.W. and Patrick, Lennon, V.A., Lindstrom, J. and Seybold, ME. (1975) J. Exp. Med. x,1365-1375. McKearn, T.J., Sarmiento, M., Weiss, A., Stuart, F.P. and Fitch, F.W. (1978) Current Topics in Microbial. Immunol. &,61-65. E. (1975) Stand. J. Immunol. Aarli, J.A., Mattsson, C. and Heilbronn, &,849-852.
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BIOCHEMKAL
AND BIOPHYSICAL RESEARCH COMMUNKATIONS
Severson, C.D. and Thompson, J.S. (1966) J. Innuunol. 95,785-789. Gomez, C.M., Richman, D.P., Berman, P.W., Burres, S.A., Arnason, B.G.W. and Fitch, F.W. (1979) Fed. Proc. (in press). Lindstrom, J. and Patrick, J. (1974) Synaptic Transmission and Neuronal Interaction, M.V.L. Bennett, ed., pp. 191-216, Raven Press, New York. Greenbury, C.L., Moore, D.H., and Nunn, L.A.C. (1963) Immunology &421-428.
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