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An antibody to eye muscle in graves' ophthalmopathy
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AN ANTIBODY I.0EYE MUSCLE IN GRAVES' OPHTHALMOPATHY. P. Kendall-Taylor, M. Holcombe, and S, Atkinson, Endocrine Unit, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP, UK. The pathogenesis of Graves' ophthalmopatby is unknown and the precise nature of its association with byperthyroidism is not clear. Previous work has suggested possible autoi~une responsiveness to retro-orbital antigens. We have developed an ELISA for detection of IgG binding to retro-orbital muscle tissue. Patients with ophthalmopathy were divided into 3 groups. In group A, with active disease, ophthalmopathic IgG (OIg) was detectable in 15124 Patients (62%); in group B, patients with active ophthalmopathy receiving treatment with prednisone, 4flO had OIg, and in group C, Patients with inactive ophthalmopathy, only 1 of 9 was positive. No IgG binding occurred in 40 healthy subjects. Of 23 patients with auto~~une or nodular thyroid disease OIg binding was seen in one. OIg did not correlate with TBII, TgAb or microsomal antibody. The OIg did not interact with thyroid tissue. We conclude that Graves' ophthalmopathy is an entity distinct from autoi~une thyroid disease. A specific OIg is present in serum of patients with ophthalmopathy, which shows some correlation with the severity of the condition, but not with thyroid disease or thyroid antibodies.
LIO.
ASSAY ANALYSIS OF NONSPECIFIC FACTIONS IN ENZ~~-LINED ~~NOS~~ENT TESTING FOR HUMAN EYE KUSCLE SPECIFIC ANTIBODIES IN GRAVES' OPHT~I~MOPA~~Y AND HASHIMOTO'S THYROIDITIS. H. Sikorska*, A. Miller and J.R. Wall, Dept. of Medicine, McGill University, The Montreal.General Hospital Research Institute, Montreal, Quebec, Canada. In an attempt to identify and subsequently isolate an autoantigenfs) in ophthalmopathy associated with thyroid disorders, normal human and pig eye muscle tissue separated into cytosol and membrane pellet, was assayed against Graves' disease and Hashimoto's thyroiditis patients, as well as normal subjects, sera in ELISA, using horseradish peroxidase conjugated anti-human IgG. When CHAPS0 (a zwitterionic detergent)-solubilisedhuman eye muscle membranes were used as antigen in ELISA, and results expressed as 0-D. corrected for the background, only 18% of patients with Graves' ophthalmopathy were found positive while as many as 50% of patients with Hashimoto's thyroiditis with no ophthalmopathy showed reactivity above upper limit of normal. However, all human tfssue preparations (liver, skeletal muscle, thyroid, connective tissue) tested for autoantigenicity in ELISA, were found to have high nonspecific activity in the absence of sera. When pig tissue was used instead of human, nonspecific binding was not detected. This nonspecific activity was reduced by 50% by pretreatment of the tissue with anti-human IgG and by 60-80% with mercaptoeth~oI, but remained unchanged after anti-human IgM pretreatment. The immunodetection of blotted human tissue with anti-human IgG confirmed that human membrane preparations contain KgG antibody capable of reacting nonspecifically with the antiserum and giving false positives in ELISA. Other methods of reducing nonspecific binding are being investigated.
AN ANTIBODY I.0EYE MUSCLE IN GRAVES' OPHTHALMOPATHY. P. Kendall-Taylor, M. Holcombe, and S, Atkinson, Endocrine Unit, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP, UK. The pathogenesis of Graves' ophthalmopatby is unknown and the precise nature of its association with byperthyroidism is not clear. Previous work has suggested possible autoi~une responsiveness to retro-orbital antigens. We have developed an ELISA for detection of IgG binding to retro-orbital muscle tissue. Patients with ophthalmopathy were divided into 3 groups. In group A, with active disease, ophthalmopathic IgG (OIg) was detectable in 15124 Patients (62%); in group B, patients with active ophthalmopathy receiving treatment with prednisone, 4flO had OIg, and in group C, Patients with inactive ophthalmopathy, only 1 of 9 was positive. No IgG binding occurred in 40 healthy subjects. Of 23 patients with auto~~une or nodular thyroid disease OIg binding was seen in one. OIg did not correlate with TBII, TgAb or microsomal antibody. The OIg did not interact with thyroid tissue. We conclude that Graves' ophthalmopathy is an entity distinct from autoi~une thyroid disease. A specific OIg is present in serum of patients with ophthalmopathy, which shows some correlation with the severity of the condition, but not with thyroid disease or thyroid antibodies.
LIO.
ASSAY ANALYSIS OF NONSPECIFIC FACTIONS IN ENZ~~-LINED ~~NOS~~ENT TESTING FOR HUMAN EYE KUSCLE SPECIFIC ANTIBODIES IN GRAVES' OPHT~I~MOPA~~Y AND HASHIMOTO'S THYROIDITIS. H. Sikorska*, A. Miller and J.R. Wall, Dept. of Medicine, McGill University, The Montreal.General Hospital Research Institute, Montreal, Quebec, Canada. In an attempt to identify and subsequently isolate an autoantigenfs) in ophthalmopathy associated with thyroid disorders, normal human and pig eye muscle tissue separated into cytosol and membrane pellet, was assayed against Graves' disease and Hashimoto's thyroiditis patients, as well as normal subjects, sera in ELISA, using horseradish peroxidase conjugated anti-human IgG. When CHAPS0 (a zwitterionic detergent)-solubilisedhuman eye muscle membranes were used as antigen in ELISA, and results expressed as 0-D. corrected for the background, only 18% of patients with Graves' ophthalmopathy were found positive while as many as 50% of patients with Hashimoto's thyroiditis with no ophthalmopathy showed reactivity above upper limit of normal. However, all human tfssue preparations (liver, skeletal muscle, thyroid, connective tissue) tested for autoantigenicity in ELISA, were found to have high nonspecific activity in the absence of sera. When pig tissue was used instead of human, nonspecific binding was not detected. This nonspecific activity was reduced by 50% by pretreatment of the tissue with anti-human IgG and by 60-80% with mercaptoeth~oI, but remained unchanged after anti-human IgM pretreatment. The immunodetection of blotted human tissue with anti-human IgG confirmed that human membrane preparations contain KgG antibody capable of reacting nonspecifically with the antiserum and giving false positives in ELISA. Other methods of reducing nonspecific binding are being investigated.