Clinical Chemistry
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 48: 549-554, 2002;
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rícny, J.
Right arrow Articles by Vincent, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rícny, J.
Right arrow Articles by Vincent, A.
Related Collections
Right arrow Clinical Immunology
(Clinical Chemistry. 2002;48:549-554.)
© 2002 American Association for Clinical Chemistry, Inc.

Determination of Anti-Acetylcholine Receptor Antibodies in Myasthenic Patients by Use of Time-resolved Fluorescence

Jan Rícny1a, Libuse Simková2 and Angela Vincent3

1 Institute of Physiology, Academy of Sciences of Czech Republic, Vídeòská 1083, 142 20 Prague, Czech Republic.

2 Neurologic Clinic, 1st Medical Faculty of Charles University, Kateøinská 30, 120 00 Prague, Czech Republic.

3 Neurosciences Group, Department of Clinical Neurology, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom.

aAuthor for correspondence. Fax 420-2-4752488; e-mail ricny{at}biomed.cas.cz.

Background: Autoantibodies against nicotinic acetylcholine receptor (nAChR) in myasthenia gravis (MG) patients are usually detected by radioimmunoprecipitation assays using extracted acetylcholine receptors labeled irreversibly with 125I-{alpha}-bungarotoxin ({alpha}-BuTx). To provide a nonradioactive immunoassay, we established an assay using nAChRs labeled with Eu3+-{alpha}-cobratoxin ({alpha}-CTx).

Methods: We derivatized {alpha}-CTx with a diethylenetriaminepentaacetate moiety and formed a complex with Eu3+. The complex was purified by HPLC, and the fractions were tested for binding to Torpedo and human nAChRs. The most active fractions were used to label nAChRs for the immunoprecipitation assay, and the bound Eu3+ was quantified by time-resolved fluorescence.

Results: Eu3+-labeled {alpha}-CTx competed with 125I-{alpha}-BuTx for binding to Torpedo nAChRs and saturated the binding sites of human nAChRs, with a Kd of 7.2 x 10-9 mol/L. Results of the immunoassay performed with Eu3+-labeled {alpha}-CTx were similar to those obtained with 125I-{alpha}-BuTx, with a slightly higher limit of detection [0.3 nmol/L (n = 6) vs ~0.1 nmol/L for isotopic assay]. None of 34 negative sera tested (16 healthy controls, 10 patients with nonmyasthenia-related disease, 8 patients seronegative for MG) gave a value >0.3 nmol/L. Of the 35 positive myasthenic sera (with antibody values, previously determined by isotopic assay, of 0.4–1290 nmol/L) compared in the two assays, 32 tested positive with the Eu3+ assay. Linear regression analysis yielded the equation: y = 1.035x - 0.013 nmol/L; Sy|x = 0.172 nmol/L; r2 = 0.977.

Conclusions: The new time-resolved fluorescence method for quantification of antibodies to nAChRs in MG patients provides a performance similar to that of the widely used isotopic assay and could be used in laboratories with restricted use of isotopes.






HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2002 by the American Association for Clinical Chemistry.