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Cloning and characterization of a lambert-eaton myasthenic syndrome antigen.

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Cloning and Characterization
of a LarGbert-Eaton Myasthenic
Syndrome Antigen
Myrna R. Rosenfeld, MD, PhD,*I- Ellen Wong, BA,? Josep Dalmau, MD," Geoff Manley, BA,?
Jerome B. Posner, MD,* Emanuele Sher, MD,S and Henry M. Furneaw, PhDI~
Lambert-Eaton myasthenic syndrome is a paraneoplastic neuromuscular disorder in which an immune response directed against a small-cell lung tumor crossreacts with antigens in the neuromuscular junction. To isolate and characterize the antigens, we screened a human fetal brain expression library with a high-titer serum from a patient with
Lambert-Eaton myasthenic syndrome. This screening resulted in the isolation of a complementary DNA clone encoding
an antigen we call myasthenic syndrome antigen B (MysB). Approximately 43% (3 of 7) of Lambert-Eaton myasthenic
syndrome sera specifically recognized MysB fusion protein, whereas none of 34 control sera did. The predicted amino
acid sequence of this clone shows a high degree of homology to the f3 subunit of calcium channel complexes. The
MysB pre-messenger RNA is alternatively spliced to yield 3 forms of the protein differing in the domain between
two highly conserved a-helical segments.
Rosenfeld MR, Wong E, Dalmau J, Manley G, Posner JB, Sher E, Furneaux HM. Cloning and characterization of a Lambert-Eaton myasthenic syndrome antigen. Ann Neurol 1993;33:113-120
Paraneoplastic neurological syndromes are disorders of
nervous system function that occur in association with
systemic cancers, particularly small-cell lung cancer. In
many of these syndromes, it is thought that neurological dysfunction arises from an immune response to the
tumor, which crossreacts with similar antigens in the
nervous system 11). Lambert-Eaton myasthenic syndrome is the only example of these syndromes in
which an immune response has been shown to be the
pathogenic agent by transfer of the disease to animals
[Z-41. First reported in 1953 by Anderson and colleagues 151, who noted muscle weakness in a patient with small-cell lung cancer, the syndrome was
named after Lambert and Eaton, who described the
electrophysiological abnormalities [6]. Further analysis
showed that the neurological deficits were the result
of insufficient presynaptic release of acetylcholine 171.
Structural and morphological analysis of affected muscle demonstrated decrease and disorganization of presynaptic active zone particles believed to be voltagegated Ca" channels 181. These observations suggested
that the deficit resided in the presynaptic voltage-gated
calcium channels, which control acetylcholine release
{9] in the neuromuscular junction.
Attention then turned to the way in which small-cell
lung tumors might cause this neuromuscular block. Ex-
tracts from small-cell lung tumors were reported to
contain a neuroactive substance that inhibited neuromuscular transmission, but these initial observations
were not confirmed [lo]. An immune etiology was first
suggested by Lang and associates [2] and is strongly
supported by all available evidence. Mice were injected
with immunoglobulin from the sera of patients with
Lambert-Eaton myasthenic syndrome. The neuromuscular deficit typical of the Lambert-Eaton syndrome
developed in these mice, in contrast to mice injected
with control immunoglobulin G (IgG). Analysis of
miniature endplate potentials in the affected mice
strongly suggested that the IgG was an irreversible
Ca2+ antagonist. Subsequently, Roberts and coworkers { 1 11 showed that IgG from patients with Lambert-Eaton myasthenic syndrome inhibited Ca2+ flux
in vitro. Although it was clear that Ca2+ channel function was inhibited, these experiments could not establish whether the Ca2+ channel was the antigenic target
or whether the IgG inhibited another system that regulated the Ca2+ channel complex.
More recently, Sher and colleagues [12) showed that
Lambert-Eaton sera could immunoprecipitate Ca2
channel complexes prelabeled with 1125-o-conotoxin.
o-Conotoxin binds a specific subtype of voltage-gated
calcium channel expressed in both neurons and small-
From the 'Department of Neurology, and the ?Laboratory of Molecular Neuro-oncology, Memorial Sloan-Kettering Cancer Center,
New York, NY, and Konsigho nazionale delle Richerche, Centro
per lo Studio della Farmacologia delle Infrastructture Cellulari, Milano, Italy.
Address correspondence to Dr Furneaux, Laboratory of Molecular
Neuro-oncology, Memorial Sloan-Kettering Cancer Center, 1275
York Ave, New York, NY 10021.
Received Sep 30, 1992, and in revised form Oct 22. Accepted for
publication Oct 22, 1992.
Copyright 0 1793 by the American Neurological Association 113
cell lung cancer cells {13, 141. These data suggested
that a subunit of the Car+ channel complex or a tightly
associated protein was the target antigen. Furthermore,
they suggested that the immune response was provoked by the expression of this Ca2' channel in smallcell lung tumors [lS]. Direct efforts to identify the
antigen by Western blot analysis have proved unsuccessful [16}.
To isolate and characterize Lambert-Eaton myasthenic syndrome antigens, we screened a human fetal
brain expression library using the serum of a patient
with Lambert-Eaton myasthenic syndrome that contained an unusually high concentration of antibodies
against voltage-gated calcium channels, as determined
by the w-conotoxin assay El 71. This screening resulted
in the isolation of 2 independent but related complementary DNA (cDNA) clones encoding antigens,
which we call myasthenic syndrome antigen A and B
(MysA, MysB). Fusion proteins derived from these
clones were specifically recognized by sera from patients with Lambert-Eaton myasthenic syndrome. T h e
predicted amino acid sequence of these clones shows
high homology to the p subunit of Ca2+ channel complexes [l 8-20].
Experimental Procedures
Screening of A Expression Libraries
A AZAP I1 fetal brain library (Stratagene) was screened at a
density of 5 x lo4p f d l 5 0 mm plate of Escherichia coli XL1Blue. After a 6-hour incubation at 37"C, plates were overlaid
with filters soaked in 10 mmoVL isopropyl P-D-thiogdactopyranoside (IPTG) and incubated for 12 hours at 37°C. Filters were removed; blocked with 5% Blotto; incubated with
the serum from patients with Lambert-Eaton myasthenic syndrome (10 pg/mL) for 2 hours at room temperature; washed
in 50 mrnol/LTris (pH, 7.4), 100 mmoULNaCI,O.2% Triton
(TBST buffer); incubated at room temperature with I'25 Protein A (0.1 FCiimL) for 1 hour; washed with TBST; dried;
and exposed to XARS film overnight at - 70°C. Clones giving positive results were purified by several rounds of antibody screening until a yield of 100% positive plaques was
obtained and then subcloned in XL1-Blue using the in vivo
excision phage rescue protocol (Stratagene).
Isolation of Overlapping cDNA Clones
The EcoRI insert from pMysB 1 was isolated and labeled with
c@*P]dCTP [21]. This labeled DNA was used to screen
the total brain AZAP I1 library. Nitrocellulose filters were
overlaid on 150 mm plates for 4 minutes; washed at room
temperature for 4 minutes in 1 rnol NaCy0.5 rnol NaOH, 2
mol NaCU1 rnol Tris (pH, 6.8), and 2 x SSC (1 x SSC =
0.15 rnol NaCVO. 15 rnol sodium citrate); then vacuum dried
for 2 hours at 80°C. Filters were then incubated with the
EcoRI insert (1 x lo6 c p d m l ) in 5 x SSC containing 1x
Denharts, 50 ~ g / m Ldenatured salmon sperm DNA, and
50% formamide for 12 hours at 42°C. Filters were then
washed and autoradiographed. Two clones, pMysB2 and
pMysB3, with additional 5' and 3' sequences, were isolated,
114 Annals of Neurology
Vol 33 N o 1 January 1993
purified, and converted to plasmids by phage rescue, as described.
Western Blot Analysis
Fusion protein and E. coli protein extract (pBS) were obtained by growing an individual colony to an optical density
of 0.6 and inducing with 10 mmoVL IPTG for 1 hour at
37°C. Cells were isolated by centrifugation and lysed by resuspension in 2% SDS and 50 mrnoVL HEPES (pH, 7.0).
Lysates (200 pg) were resolved by 10% SDS/polyacrylamide
gel electrophoresis and transferred to nitrocellulose C221.
The nitrocellulose was blocked with 3% bovine serum albumin and incubated with the indicated amount of serum for
2 hours at room temperature, washed with TBST, incubated
with I*25Protein A for 30 minutes, dried, and exposed to
XARS film at -70°C.
Reverse Transcriptase-PolymeraseChain Reaction
Analysis of MysB Transcripts
Poly(A)+ RNA (50 ng) from various human tissues (Clontech) was incubated with reverse transcriptase (2.5 U/pL) for
30 minutes at 42°C in a total reaction volume of 20 pL
containing the following: 1 mmoVL each dNTP, 1 UIp1 ribonuclease I inhibitor, and 2.5 pmoYL random hexamer primers. Reverse transcriptase (RT) reactions were terminated by
incubation at 99°C for 5 minutes and used directly for subsequent polymerase chain reaction (PCR) amplification of specific cDNAs.
One-twentieth of the RT reaction product was added to a
PCR (50 FL final value; sample overlaid with 50 pL paraffin
oil), which contained 1 x PCR buffer (50 mmol/L Tris [pH,
9.5), 1.5 mmoUL MgCI,, 20 mmol/L ammonium sulfate); 0.2
mmoVL each dNTP; 5 pCi C X [ ~ ~ P ] ~ C0.5
T P ;KmoUL each
5' and 3' primer (CAGTGCGGCCCATGAAGA, CGGAGGAGTGTGCTCTGT) for MysB; and 1U taq polymerase
(Perkin-Elmer Cetus). PCR analyses were performed in an
automated DNA clonal cycler (Ericomp) with the following temperature profile: 1 minute at 90"C, 35 cycles of 30
seconds at 53"C, 1 minute at 72"C, and 1 minute at 92°C; 2
minutes at 55°C; and 10 minutes at 72°C. One-tenth of the
PCR product was electrophoresed in 6% acrylamide gel, and
the PCR fragments were analyzed by autoradiography. PCR
reaction product was subcloned using an A-T tailed vector
(pT7Blue(U) T-vector; Novagen). Colonies were assayed by
PCR with vector primers to establish the presence and size
of inserts. Inserts were then sequenced using the dideoxy
sequencing methods [231 and MysB oligonucleotide primers.
D N A Sepence Analysis
Sequence analysis was performed using the dideoxy terrnination method f231 with Sequenase 2.0 (United States
Biochemical). Double-stranded DNA was purified using the
Qiagen plasmid midi-prep system and sequenced on both
strands. Single-stranded DNA was prepared using a modified
single-strand rescue protocol (Stratagene). Internal oligonucleotide primers, as well as SK and KS primers, were used.
lsolation of cDNA Clones
Screening of 2 X lo6 phage from a hZAP human fetal
brain library resulted in the isolation of 2 positive
Fig 1. Reactivity of phage clones with sera. Phage clone 8FZ6.6
was plated and reacted with (A) n o m l human serum and (8)
Lambert-Eaton myasthenic syndrome serum. An iwelevant phage
clone (selected at random) was also plated and reacted with (C)
normal human serum and (0)Lambert-Eaton myasthenic syndrome serum.
clones (8FZ6.6 and 1FZ4.5). Neither of the clones
reacted with normal human serum. Figure 1 shows the
reactivity of clone 8FZ6.6 with normal human sera (Fig
1A) and Lambert-Eaton sera (Fig 1B). There was no
difference in the reactivity of normal human serum
(Fig 1C) and Lambert-Eaton serum on incubation with
an irrelevant phage clone (Fig 1D). Both clones were
subcloned into pBluescript using the phage excision
protocol and analyzed by restriction enzyme digestion.
Clone 8FZ6.6 contained an R 1 insert of 424 nucleotides (pMysBl), whereas clone 1FZ4.5 contained an
R1 insert of 2,419 nucleotides (pMysA1). Southern
blot analysis showed that these inserts did not anneal
to each other under high-stringency wash conditions
(0.1 x SSC at 65°C). We concluded that these two
clones encode two gene products significantly different
in nucleotide sequences but similar enough at the
amino acid level to be recognized by the same sera.
This conclusion has been confirmed by nucleic acid
sequence analysis. We called the gene product encoded
by 1FZ4.5, MysA, and the gene product encoded by
8FZ6.6, MysB. We focus exclusively on MysB herein.
Specific Recognition of pMysB cDNA Clone by
Lambert-Eaton Sera
The reactivity of h b e r t - E a t o n sera and various negative control sera was established by Western blot analysis of the fusion protein encoded by pMysB1. LambertEaton sera react with a fusion protein of 35 kd and a
minor protolytic fragment of 30 kd in extracts of
pMysB1 (Fig 2). No reactivity was observed with extracts of E . coli. Normal human serum reacted weakly
Fig 2. Western blot analysis of MysB fusion protein. Preparations of Escherichia coli protein (lanes I and 3) and MysB fusion protein (lanes 2 and 4) reacted with normal human serum
and Lambert-Eaton myasthenic syndrome serum. Both sera were
tested at 112,000.
with a protein band of 33 kd that was present in extracts of E. coli and pMysB1. We examined the specificity of this antigen by examining its reactivity with 7
sera samples from patients who had been diagnosed as
having Lambert-Eaton syndrome by electrophysiological criteria. Figure 3 shows that 3 of the 7 sera reacted
with pMysB1 fusion protein. The other sera only reacted with E . coli proteins, as evidenced by their similar
reactivity with extracts of pBS (data not shown). None
of 34 negative control sera were reactive. These control sera included sera from 5 normal individuals, 7
patients with small-cell lung cancer, 10 with cancer and
other antibody-associated paraneoplastic syndromes, 6
with systemic lupus erthyematosus, 2 with Sjogren’s
syndrome, and 1 each with myasthenia gravis, multiple
sclerosis, epilepsy, and migraine. The characteristics of
the 7 Lambert-Eaton sera samples are summarized in
the Table. There is a clear correlation between reactivity with pMysB1 and reactivity with u-conotoxinlabeled calcium channel complexes.
Complete Strzlctzrre and Sequence Analysis of MysB
pMysB1 was sequenced on both strands using T3, T7,
and internal oligonucleotide primers. The insert encodes a predicted protein of 142 amino acids, with a
predicted molecular weight of 15.6 kd. The observed
molecular weight of 35 kd results from an in-frame
Rosenfeld et al: Lambert-Eaton Myasthenic Syndrome Antigen
cDNA clones encoding MysB
Fig 4. Structure of cDNA clones encoding MysB.
Fig 3 . MysB fusion protein is selectively recognized by sera from
patients witb Lambert-Eaton myasthenic syndrome. MysB f u sion protein was run on a preparative (curtain) 10% SDS gel.
Protein was transfewed to nitrocellulose {22} and reacted with
(B) no sera, (N)normal human serum, and (1-7) sera from patients with Lumbert-Eaton myasthenic syndrome.
Characteristics of Lumbert-Eaton Sera Samples
Code No.
s #7
s #8
G5 718
NHL = non-Hodgkins lymphoma; SCLC = small-cell lung cancer;
NCD = not clinically detectable; ND = not done.
insertion at both the N- and C-termini of P-galactosidase.
We completed the sequence of MysB. Eleven overlapping cDNAs were isolated by screening a fetal brain
library with the EcoRI insert of pMysB1. Two of these
cDNA clones (pMysB2 and pMysB3) comprise the
complete structure of MysB (Fig 4). All cDNAs were
sequenced on both strands, and the complete sequence
is shown in Figure 5 . There is a long, open reading
frame encoding a predicted protein of 566 amino acids
starting at an AUG at position 61 and terminating at
position 1,760. The rare codon usage algorithm indi-
116 Annals of Neurology Vol 33 No 1 January 1993
cates that the sequences preceding the AUG at position 61 are unlikely to code for protein. There is a
polyA tail that is preceded by a consensus polyA addition sequence. The predicted amino acid sequence was
used in a homology search of Genpept and Swiss-Prot
data bases and revealed striking homology (76.6%
identity over 461 amino acids) with the p subunits of
Ca2+channel complexes. Figure 6 shows the homology
alignment of MysB with p subunits cloned from rabbit
skeletal muscle 1181, rat brain 1191, and human hippocampus 120). This alignment reveals two highly conserved a-helical segments connected by a more variable domain, which, in the case of the human p2
subunit, may be alternatively spliced 120). All 4 proteins diverge considerably at the C-terminus. Analysis
of functional domains using the Blast program [241
revealed the presence of an SH3 (Src homology 3)
domain (residues 114-174) in the first highly conserved segment. The function of SH3 domains is not
well understood, but its presence may imply that the
p subunit is involved in a cascade of proteins regulated
by phosphorylation C253.
We sequenced the 2,419 nucleotide insert of
pMysA1. pMysA1 contains an open reading frame of
448 amino acids and a 3' untranslated region of 1,075
nucleotides. The predicted amino acid sequence shows
high homology (70% identity over 448 amino acids)
to the p subunit gene family. O n Western blot analysis,
LEMS sera #1 reacted with a fusion protein of 55 kd.
The level of reactivity was significantly less than that
exhibited by pMysB1. For this reason, the reactivity of
pMysA1 with other sera was not determined. We were
also unable to isolate a full-length MysA cDNA clone
and have not characterized further this gene product.
Expression and Alternative Splicing of MysB
Messenger RNA
We used the RT-PCR assay to detect MysB messenger
RNA (mRNA) in various human tissues. Primers that
were specific for MysB (18-oligomers) and spanned
the potential alternatively spliced region found in the
Fig 5 . Nucleotide sequence and predicted amino acid sequence of
human p2 subunit were used C201. An RT-dependent
product of 447 nucleotides was observed on analysis
of liver, muscle, lung, brain, and HeLa cell RNA. We
concluded that MysB is probably expressed in all human tissues; unfortunately, we could not establish the
expression of MysB in tumor tissue from a patient with
Lambert-Eaton myasthenic syndrome because this material was not available. In addition, minor products of
375 and 333 nucleotides were also found (Fig 7).
Subcloning and sequence analysis showed that the
PCR products correspond to 3 alternatively spliced
MysB mRNAs. These 3 transcripts give rise to 3
forms of MysB, which differ in the domain that connects the 2 highly conserved a-helical segments (see
Fig 6). The smallest transcript encodes a small domain
(AKQKQKS), which is identical to the domains found
in the rat brain @ subunit and one of the isoforms
of the human hippocampal @ 2 subunit. The other 2
transcripts encode domains that are very different from
the corresponding ones found in all the other cloned
p subunits. We can only speculate on the function of
this alternative splice. One possibility is that alterations
in this domain may allow the two a-helical segments
to adopt a crucial conformation required for productive
interaction with a particular a1 subunit. Thus, the alter-
native splice may regulate the association of @ subunits
with a specific class of channel complexes.
Using serum from a patient with Lambert-Eaton myasthenic syndrome, we characterized human cDNA
clones that are highly homologous to the p subunit of
the voltage-gated calcium channel. There is increasing
evidence that there may be a large family of @ subunit
proteins associated with the different pharmacological
types of Ca2+ channels 1261. The human antigen that
we describe, MysB, appears to be a novel member of
this gene family.
The diagnosis of Lambert-Eaton myasthenic syndrome is currently based on clinical and electrophysiological criteria 1277. Electromyographic findings of all
patients show a reduced amplitude of the compound
muscle action potential, which increases progressively
during repetitive supramaximal nerve stimulation [7}.
All patients with Iambert-Eaton myasthenic syndrome
who are tested have IgG that inhibits CaZ+ flux in
small-cell lung cancer cell lines, and the magnitude of
inhibition correlates with the severity of LambertEaton myasthenic syndrome symptoms C281. Currently, the only assay available for detection of antibodies in Iamben-Eaton myasthenic syndrome sera is
precipitation of w-conotoxin-labeled voltage-gated calcium channels Cl2, 17, 291. However, not all patients
Rosenfeld et al: Lambert-Eaton Myasthenic Syndrome Antigen
u u
"*" ~-.......513
N H P P G r U G T L I A L S R Q D T F O A ~ P ~ S R N S W T E P G D S C V D n Y 5C7 1
..TSLRIINLSF ..............................................
ffiLE=.. .
F i g 6. (A)Homology alignment of MysB t o /3 subunits from rat
brain (brain), human hippocampus (/32),and rabbit skeletal
muscle (mude). Homology alignment was perfamed using GeneWorks sequence analysis software. (B) Conserved domains of p
subunits. The shaded regions indicate highly conserved
(>SO% identity) domains. The dotted line indicates the region that is alternatively spliced. The heavily shaded region
(SH3) indicated the Sarc homology 3-like domain.
Fig 7 . Analysis of alternately spliced MysB transcripts. The
left panel indicates human brain mRNA assayed in the absence
( - ) and presence ( -k ) of reverse transcriptase (RT). Deduced
amino acid sequence of each transcript is shown t o the right. Alternatively spliced sequences are highlighted. The size of the
PCR products is noted to the left.
118 Annals of Neurology Vol 33 No 1 January 1993
with characteristic electrophysiological findings have
detectable levels of these antibodies. Similarly, in our
study, all the sera from patients with Lambert-Eaton
myasthenic syndrome did not react with MysB fusion
protein. Thus, reactivity with MysB fusion protein is
specific for a subtype of patients with Lambert-Eaton
myasthenic syndrome.
There are at least 3 possible reasons why all patients
with Lambert-Eaton myasthenic syndrome do not identify MysB fusion protein: (1)the current assays are not
sensitive enough to detect low concentrations of anti-P
subunit antibodies; ( 2 ) although all patients with Lambert-Eaton myasthenic syndrome have an immunemediated defect of calcium channel function, the target
antigens to which the IgG antibodies are directed may
be heterogeneous; and (3) although antibodies directed
against MysB are specific for some patients with Lambert-Eaton myasthenic syndrome, they are not relevant
for the pathogenesis of the disease.
During preparation of this article, other investigators
1301 reported the association of synaptotagmin with
Ca2' channels and suggested that it may also be a Lambert-Eaton myasthenic syndrome antigen. In these
studies, reactivity of Lambert-Eaton syndrome sera
with recombinant (i.e., purified) synaptotagmin was not
demonstrated. Identity of the antigen rested on the
molecular weight of the protein characterized by Western blot analysis of partially purified calcium channel
complexes. It is important to note that synaptotagmin
and the (3 subunit are of similar molecular weight. It is
also possible that reactivity with synaptotagmin may
define the subset of E M S sera that do react with the
p subunit.
For patients with Lambert-Eaton myasthenic syndrome with anti-p subunit antibodies, disruption of
neuromuscular function by Lambert-Eaton myasthenic
syndrome sera may be due to binding of IgG to the
p subunit. The skeletal muscle voltage-gated calcium
channel consists of 5 subunits ( a l , a2, (3, y, and 8)
1311. The a1 subunit is the central ion channel component, which binds classic calcium channel antagonists
and is highly homologous to the a subunits of other
voltage-gated ion channels 1323. The (3 subunit accelerates both activation and inactivation of the calcium
channel current and increases the number of calcium
antagonist binding sites after transfection into LCa. 11
cells 1331. Similar modulation of the (3 subunit on voltage-gated calcium channel expression in Xenopus
oocytes has been obtained after injection of a cloned
neuronal a1 subunit in conjunction with the cloned
a2 subunit [34].
Lambert-Eaton myasthenic syndrome antibodies inhibit voltage-gated calcium channels by inducing internalization and subsequent degradation 1351. Current
models of the Ca2+ channel complex place the f3 subunit inside the cell membrane [361. Accordingly, it is
difficult to envision that an anti+ subunit IgG could
induce internalization; however, there is no direct evidence of the topology of the p subunit, and there is a
possibility that some domains may be extracellular by
virtue of associations with the a1 subunit. Immunization of animals with these recombinant proteins may
determine the role of these antigens in the pathogenesis of the disease and could provide a Lambert-Eaton
animal model that so far has only been obtained with
the passive transfer of Iambert-Eaton myasthenic syndrome serum or IgG 12, 3, 373.
Why do patients with Lambert-Eaton myasthenic
syndrome mount an immune response against subunits? In patients with associated small-cell lung cancer
(60%), the hypothesis we favor is that the amino acid
sequence of the f3 subunit expressed by the tumor is
altered and therefore perceived as foreign. There is
precedence for this hypothesis: Patients with cancer
mount an immune response against mutant forms of
the protooncogene p53 expressed in breast tumors
138,391. This hypothesis can now be tested by comparing the structure of the f3 subunit in small-cell lung
Although neurological paraneoplastic syndromes are
rare, they may provide general clues to development
of an antitumor immune response 140-421. Perhaps
many autoimmune diseases (e.g., myasthenia gravis)
are triggered by aberrant expression of the autoantigen
in small benign tumors. For clinicians, cloning of the
recombinant antigens associated with some paraneoplastic diseases has provided unambiguous clinical assays not only to diagnose the paraneoplastic disorder,
but also to direct the search for the tumor most commonly involved 143-461.
This work was supported by ACS PDT B9 (H.F.), and N I H
NS26064 (J.B.P.). M. Rosenfeld is a Charles A. Dana Foundation
Fellow and is supported in part by the National Institutes of Health
Clinical Scholars Training grant CA09512. J. Dalmau is supported
by agrant from FISS (Spain). G. Manley is a medical scientist training
program fellow. This work was also supported by an industrial agreement with Genica Pharmaceutical Corporation.
We are very grateful to Dr T. Vollmer, D r P. Clouston, and Dr F.
Graus for providing us with Lambert-Eaton myasthenic syndrome
sera; and Dr Newsom-Davis and Ms I. Johnston, who assayed the
serum for o-conotoxin-voltage-gatedcalcium channel reactivity. We
thank B. Nevins for his patience in preparing the manuscript.
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cloning, lambert, syndrome, antigen, myasthenia, eaton, characterization
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