title: Recombinant expression of the AChR-alpha1 subunit for the detection of conformation-dependent epitopes in Myasthenia Gravis.
abstract: Detection of autoantibodies associated with neurological disease typically involves immunoprecipitation of radioactively labeled native proteins. We explored whether single receptor subunits, fused to Renilla luciferase (Ruc), could detect patient autoantibodies in Luciferase Immunoprecipitation Systems. Myasthenia Gravis patient sera were tested for conformational autoantibodies to only the α1-subunit of the nicotinic acetylcholine receptor (AChR). Using a panel of 10 AChR-α1 fragments, AChR-α1-Δ5-Ruc demonstrated the highest immunoreactivity with a conformational-specific antibody and the highest sensitivity in a pilot cohort. Testing a larger cohort with AChR-α1-Δ5-Ruc demonstrated 21% sensitivity and 97% specificity. A point mutation within Ruc increased the diagnostic performance of AChR-α1-Δ5 (32% sensitivity, 97% specificity). The (125)I-α-bungarotoxin multi-subunit AChR assay demonstrated 63% sensitivity and 97% specificity. These findings highlight the difficulty in detecting Myasthenia Gravis conformational epitopes across assay formats and lay the foundation for detecting autoantibodies to defined recombinant chains of the AChR and potentially other neurotransmitter receptors.
authors.affiliation: Neurobiology and Pain Therapeutics Section, Laboratory of Sensory Biology, National Institute of Dental and Craniofacial Research, 49 Convent Drive, Bethesda, MD 20892-4410, United States.
authors.forename: Kathryn H
authors.initials: KH
authors.lastname: Ching
authors.forename: Peter D
authors.initials: PD
authors.lastname: Burbelo
authors.forename: Richard M
authors.initials: RM
authors.lastname: Kimball
authors.forename: Lora L
authors.initials: LL
authors.lastname: Clawson
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Corse
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Iadarola
mesh_terms: D000818:Animals; D001323:Autoantibodies / Q000097:blood*; D002038:Bungarotoxins / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009157:Myasthenia Gravis / Q000097:blood*; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D011863:Radioimmunoassay / Q000379:methods; D011978:Receptors, Nicotinic / Q000235:genetics / Q000276:immunology*; D014162:Transfection / Q000379:methods
chemical_list: D001323:Autoantibodies; D002038:Bungarotoxins; D000939:Epitopes; D011978:Receptors, Nicotinic; D008156:Luciferases