TE671 Cell-based ELISA for Anti-Acetylcholine
Receptor Antibody Determination in Myasthenia
Gravis
Diego Franciotta,
1*
Gianvito Martino,
2
Elena Brambilla,
3
Elisabetta Zardini,
1
Vera Locatelli,
1
Alessandra Bergami,
2
Carmine Tinelli,
4
Gaetano Desina,
5
and
Vittorio Cosi
6
Background: Acetylcholine receptor (AChR) from hu-
man muscles is the antigen used currently in radioim-
munoprecipitation assays (RIPAs) for the determination
of anti-AChR antibodies in the diagnosis of myasthenia
gravis (MG). Our aim was to develop and validate an
ELISA using TE671 cells as the source of AChR.
Methods: After TE671 cell homogenization, the crude
AChR extract was used for plate coating. Anti-AChR
antibodies were determined in 207 MG patients and in 77
controls.
Results: The mean intra- and interassay CVs (for two
samples with different anti-AChR antibody concentra-
tions) were 9.7% and 15.7%, respectively. Test sensitivity
and specificity, for generalized MG, were 79.5% (95%
confidence interval, 72.8 – 85.0%) and 96.1% (89.0 –99.1%).
The detection limit was 2 nmol/L. Anti-AChR antibody
concentrations from 53 MG patients, as tested with our
ELISA, showed good agreement with an RIPA with a
mean difference (SD) of 1.0 (5.6) nmol/L.
Conclusion: Our ELISA is a simple screening test for
the diagnosis of MG and enables rapid and inexpensive
patient follow-up.
© 1999 American Association for Clinical Chemistry
Circulating antibodies to the acetylcholine receptor (AChR)
7
are essentially polyclonal and heterogeneous IgG, and rec-
ognize diverse epitopes of the skeletal muscle nicotinic
AChR. These antibodies are pathogenic in myasthenia gra-
vis (MG) (1), a disease characterized by fatigability and
relative weakness of voluntary muscles. Up to 90% of
patients with generalized MG, and a lower percentage of
those with ocular MG, have detectable serum concentrations
of anti-AChR antibodies. The quantification of these anti-
bodies is currently used as a laboratory support for diagno-
sis, therapy monitoring, and follow-up in MG. Antibody
concentration is commonly determined by exploitation of
the ability of anti-AChR antibodies to immunoprecipitate-
solubilized human muscle AChR, which, in turn, is com-
plexed with
125
I-labeled -bungarotoxin (
125
I--BuTx), a po-
tent acetylcholine antagonist. The main disadvantages of
this sensitive method are the use of radioisotopes and the
limited availability of human muscles. Furthermore, the
presence of -BuTx deprives a part of the anti-AChR anti-
bodies of potential binding sites.
Various authors have proposed alternative solid-phase
immunoenzymatic methods to overcome some of the above-
mentioned limitations. One method involves the coating of
polystyrene wells directly with muscle extracts (2); another
method uses muscle-derived AChR, which in turn is cap-
tured by means of precoating with -BuTx (3) or with
anti-AChR monoclonal antibodies (4). The main drawbacks
of these methods are the paucity of AChR in relation to other
muscle proteins (2) and the inadequacy of AChR capture,
whether by -BuTx or by anti-AChR monoclonal antibodies
(3, 4). The effect of these drawbacks is a reduction in the
diagnostic sensitivity of these methods.
1
Laboratory of Neuroimmunology and
6
Division B, Istituto di Ricovero e
Cura a Carattere Scientifico, Neurological Institute ‘Mondino’, via Palestro 3,
University of Pavia, 27100 Pavia, Italy.
2
Experimental Neuroimmunotherapy and
3
Neuroimmunology Unit, De-
partment of Biotechnology, San Raffaele Scientific Institute, via Olgettina 58,
20132 Milan, Italy.
4
Unit of Biometric, Istituto di Ricovero e Cura a Carattere Scientifico,
Policlinico S. Matteo, University of Pavia, 27100 Pavia, Italy.
5
‘Casa Sollievo della Sofferenza’, 71013 San Giovanni Rotondo (FG), Italy.
*Author for correspondence. Fax 39-0382-380286; e-mail francid@
cpbim1.unipv.it.
Received June 1, 1998; accepted December 28, 1998.
7
Nonstandard abbreviations: AChR, acetylcholine receptor; MG, myasthe-
nia gravis;
125
I--BuTx,
125
I-labeled -bungarotoxin; RIPA, radioimmunopre-
cipitation assay; SLE, systemic lupus erythematosus; and PBS, phosphate-
buffered saline.
Clinical Chemistry 45:3
400 – 405 (1999)
Clinical Immunology
400