Molecular cloning of the canine nicotinic acetylcholine receptor α-subunit gene and development of the ELISA method to diagnose myasthenia gravis

We investigated the molecular structure of canine nicotinic acetylcholine receptor (AChR) α-subunit gene and developed an enzyme-linked immunosorbent assay (ELISA) as an immunological method to diagnose myasthenia gravis (MG). Canine AChR α-subunit cDNA was constructed from mRNA isolated from skeletal muscle of five dogs using the reverse transcriptase–polymerase chain reaction and its molecular structure was determined. The canine AChR α-subunit gene had 1371 base pairs encoding 457 amino acids and had a 96.1% homology to the human AChR α-subunit gene at the amino acid level. From the results of sequencing the DNA, specific antibodies to the acetylcholine binding domain of the canine AChR α-subunit were produced by immunizing rabbits with synthetic oligopeptides (α-subunit 183–200 amino acids). The specificity of the rabbit anti-oligopeptide serum was examined by Western blot analysis using an E. coli-expressed AChR α-subunit protein and an AChR α-subunit protein fraction prepared from canine skeletal muscle as an antigen. An ELISA assay was developed using oligopeptides corresponding to the binding domain to diagnose canine MG; specific antibodies were detected from two dogs with MG, one diabetic dog and two healthy dogs among 25 dogs examined. Further examinations of the ELISA using a large number of samples of clinically MG-positive and MG-negative dogs are needed to establish its usefulness in MG diagnosis.