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Molecular Cloning of the Canine Nicotinic Acetylcholine Receptor Alpha-Subunit Gene and Development of the ELISA Method to Diagnose Myasthenia Gravis

Molecular Cloning of the Canine Nicotinic Acetylcholine Receptor Alpha-Subunit Gene and Development of the ELISA Method to Diagnose Myasthenia Gravis

T Yoshioka, Y Uzuka, S Tanabe, T Sarashina, N Ishiguro

Vet Immunol Immunopathol. 1999 Dec 30;72(3-4):315-24.

PMID: 10628675

Abstract:

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.

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IAR42416765	Anti-phospho-Estrogen Receptor-α (pSer305) antibody produced in rabbit			Price

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