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

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-α-bungarotoxin (α-BuTx). To provide a nonradioactive immunoassay, we established an assay using nAChRs labeled with Eu3+-α-cobratoxin (α-CTx). Methods: We derivatized α-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 α-CTx competed with 125I-α-BuTx for binding to Torpedo nAChRs and saturated the binding sites of human nAChRs, with a Kd of 7.2 × 10−9 mol/L. Results of the immunoassay performed with Eu3+-labeled α-CTx were similar to those obtained with 125I-α-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.