Re-evaluation of eye muscle autoantibody determination in Graves' ophthalmopathy: failure to detect a specific antigen by use of enzyme-linked immunosorbent assay, indirect immunofluorescence, and immunoblotting techniques

1989 ◽  
Vol 121 (5) ◽  
pp. 643-650 ◽  
Author(s):  
E. Schifferdecker ◽  
U. Ketzler-Sasse ◽  
B. O. Boehm ◽  
H. B. Ronsheimer ◽  
W. A. Scherbaum ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Indirect Immunofluorescence ◽  
Specific Antigen ◽  
Antigenic Structure ◽  
Muscle Membrane ◽  
Enzyme Linked Immunosorbent Assay ◽  
Eye Muscle ◽  
Human Eye ◽  
Graves Ophthalmopathy ◽  
Indirect Immunofluorescence Technique

Abstract. Sera from 41 patients with Graves' ophthalmopathy were investigated for presence of autoantibodies directed against eye muscle preparations using different methods: 1. ELISA with pork eye muscle membrane preparations; 2. Immunoblotting with glycoprotein preparations from human eye muscle; 3. Indirect immunofluorescence with human eye muscle sections. The ELISA was not suitable for detection of specific immunoglobulin binding with sera from patients suffering from endocrine ophthalmopathy. Immunoblotting exhibited only nonspecific binding to some muscle proteins; it could be prevented by pre-adsorption procedures and was not different from the pattern observed with skeletal muscle as antigen. The indirect immunofluorescence technique revealed no binding of Graves' sera to human muscle sections, whereas sera containing antibodies against skeletal muscle bound to eye muscle as well. Thus far, an antigenic structure of eye muscle specific for Graves' ophthalmopathy is not detectable with the methodology used here. The possibility that retroorbital connective tissue may be the main target of the autoimmune process must be considered.

Evaluation of an enzyme-linked immunosorbent assay for the measurement of autoantibodies against eye muscle membrane antigens in Graves' ophthalmopathy

1986 ◽  
Vol 113 (4) ◽  
pp. 514-522 ◽  
Author(s):  
A. Miller ◽  
H. Sikorska ◽  
M. Salvi ◽  
J. R. Wall
Keyword(s):  
Immunosorbent Assay ◽  
Specific Binding ◽  
Normal Subjects ◽  
Muscle Membrane ◽  
Eye Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Eye Muscle ◽  
Isolation And Purification ◽  
Graves Ophthalmopathy ◽  
Membrane Antigens

Abstract. We have tested for antibodies against human and pig eye muscle membrane antigens in the serum of patients with Graves' ophthalmopathy using an enzyme-linked immunosorbent assay (ELISA). Several different membrane preparations were used as source of putative antigen including a 100 000 × g pellet, a pellet depleted of the 100 000 × g (microsome) fraction, and solubilized membranes. With eye muscle membrane pellets there were no significant differences for either serum or immunoglobulins between patients with ophthalmopathy, those with autoimmune thyroid disorders without eye disease, and normal subjects for either human or pig membranes, although tests were positive determined from the upper limit of normal in a few patients with or without eye disease. This was the case regardless of the enzyme-antibody conjugate used, the membrane protein concentration or serum or immunoglobulin dilution. Pre-absorption of tissue fractions, serum, or immunoglobulins, with red blood cells or liver powder, eye muscle membranes or skeletal muscle membranes did not significantly reduce background binding which was often very high, or enhance the difference between patients with ophthalmopathy and normal subjects. It was found that non-specific binding to the plastic surface of the microplates and/or tissue proteins, the presence, in human tissues, of blood-derived immunoglobulins which gave strong reactions in the ELISA, and variable fixation of membrane pellets to the plates were factors which made ELISA unsatisfactory when crude membrane pellets were used as antigen. When eye muscle membranes solubilized with standard agents including SDS, Triton X-100 and deoxycholine were tested, again no differences were demonstrated between patients with Graves' ophthalmopathy and normal subjects. However, when membranes were solubilized with the zwitterionic agent 'CHAPSO' approximately 20% of patients with Graves' ophthalmopathy and smaller proportions of patients with thyroid disease without apparent eye involvement had positive tests with both human and pig eye muscle. While availability of human monoclonal antibodies should soon allow the isolation and purification of soluble eye muscle membrane autoantigens for use in sensitive and specific antibody tests, the ELISA, as presently used with crude tissue fractions, appears too variable and prone to non-specific immunoglobulin-binding for routine clinical use.

Immunologically Mediated Cytotoxicity Against Human Eye Muscle and Thyroid Cells in Euthyroid and Thyrotoxic Graves’ Ophthalmopathy

Autoimmunity ◽  
1991 ◽  
Vol 9 (4) ◽  
pp. 293-300 ◽  
Author(s):  
Geraldo Medeiros-neto ◽  
Zhi-guong-zhang ◽  
Nicolau Lima ◽  
Angela Iacona ◽  
Adrienne Liberman ◽  
...  
Keyword(s):  
Thyroid Cells ◽  
Eye Muscle ◽  
Human Eye ◽  
Graves Ophthalmopathy

scholarly journals Actinobacillus actinomycetemcomitans Serotype d-Specific Antigen Contains the O Antigen of Lipopolysaccharide

2003 ◽  
Vol 71 (9) ◽  
pp. 5005-5011 ◽  
Author(s):  
Laura Lakio ◽  
Susanna Paju ◽  
Georg Alfthan ◽  
Terttu Tiirola ◽  
Sirkka Asikainen ◽  
...  
Keyword(s):  
Western Blotting ◽  
Specific Antigen ◽  
Rabbit Antiserum ◽  
Actinobacillus Actinomycetemcomitans ◽  
Antigenic Structure ◽  
Enzyme Linked Immunosorbent Assay ◽  
Affinity Column ◽  
Dependent Manner ◽  
Equal Concentration ◽  
O Antigen

ABSTRACT Actinobacillus actinomycetemcomitans is a gram-negative, facultatively anaerobic bacterium which is associated especially with aggressive forms of periodontitis. Contradictory results on the localization of the A. actinomycetemcomitans serotype-specific antigen have been reported. The aim of the present study was to characterize the A. actinomycetemcomitans serotype d-specific antigen. The antigen was isolated by affinity chromatography. The affinity column was prepared from immunoglobulin G isolated from rabbit antiserum raised against A. actinomycetemcomitans serotype d. The isolated antigen was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and silver staining, all of which revealed a ladder-like structure typical for the O antigen of lipopolysaccharide (LPS). In a displacement enzyme-linked immunosorbent assay (ELISA), the isolated antigen displaced in a concentration-dependent manner the binding of the polyclonal rabbit antiserum raised against A. actinomycetemcomitans serotype d to the competing whole-cell serotype d antigen. The isolated antigen contained LPS, and an equal concentration of LPS isolated from A. actinomycetemcomitans serotype d gave a similar displacement curve in the ELISA. In order to test the immunogenic properties of the isolated antigen, it was used to immunize a rabbit. The antiserum raised against the isolated antigen displayed specificity in Western blotting and ELISA similar to that of antibody raised against LPS isolated from A. actinomycetemcomitans serotype d. In conclusion, our results show that the A. actinomycetemcomitans serotype d-specific antigen contains the O-antigenic structure of LPS.

Immunologically Mediated Cytotoxicity against Human Eye Muscle Cells in Graves’ Ophthalmopathy*

1986 ◽  
Vol 63 (2) ◽  
pp. 316-322 ◽  
Author(s):  
PEI WEN WANG ◽  
Y. HIROMATSU ◽  
E. LARYEA ◽  
L. WOSU ◽  
J. HOW ◽  
...  
Keyword(s):  
Muscle Cells ◽  
Eye Muscle ◽  
Human Eye ◽  
Graves Ophthalmopathy

The specificity of autoantibodies in Graves' ophthalmopathy

1987 ◽  
Vol 116 (1_Suppl) ◽  
pp. S330-S333 ◽  
Author(s):  
P. Kendall-Taylor ◽  
D. Jones ◽  
S. Atkinson
Keyword(s):  
Skeletal Muscle ◽  
Autoimmune Disease ◽  
Patient Population ◽  
Disease Process ◽  
Cytoskeletal Proteins ◽  
Tsh Receptor ◽  
Eye Muscle ◽  
Graves Ophthalmopathy ◽  
Organ Specific ◽  
Hashimoto's Disease

Abstract. An autoantibody to orbital antigens is found in patients with Graves' ophthalmopathy. This autoantibody also binds to skeletal muscle antigen. Occasional positive responses have been seen in patients with Hashimoto's disease and with non-organ-specific autoimmune disease. Eye muscle has a higher innervation ratio than skeletal muscle and may be of different embryological origin, but it is nevertheless closely related to skeletal muscle and it is perhaps not surprising that the antibody should interact with both these types of muscle. What remains quite unexplained, is why the disease process apparently affects eye muscle only. The antigen appears to be distinct from thyroidal antigens, but further work with a larger patient population is necessary before one can be certain of this. To date it has been possible to exclude the cytoskeletal proteins, myosin and actin, and also the TSH receptor as possible candidates for the responsible antigens. Further studies are in progress to elucidate the nature of this muscle antigen.

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