Molecular cloning of the canine nicotinic acetylcholine receptor alpha-subunit gene and development of the ELISA method to diagnose myasthenia gravis.
We investigated the molecular structure of canine nicotinic acetylcholine receptor (AChR) alpha-subunit gene and developed an enzyme-linked immunosorbent assay (ELISA) as an immunological method to diagnose myasthenia gravis (MG). Canine AChR alpha-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 alpha-subunit gene had 1371 base pairs encoding 457 amino acids and had a 96.1% homology to the human AChR alpha-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 alpha-subunit were produced by immunizing rabbits with synthetic oligopeptides (alpha-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 alpha-subunit protein and an AChR alpha-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.
Yoshioka
T
T
Department of Veterinary Internal Medicine, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan.
Uzuka
Y
Y
Tanabe
S
S
Sarashina
T
T
Ishiguro
N
N
D000109:Acetylcholine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001323:Autoantibodies / Q000032:analysis; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D006801:Humans; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000737:chemistry; D009157:Myasthenia Gravis / Q000235:genetics / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011817:Rabbits; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000276:immunology; D017422:Sequence Analysis, DNA
D001323:Autoantibodies; D011978:Receptors, Nicotinic; D000109:Acetylcholine