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.

scholarly journals Cloning and Characterization of the Novel Thyroid and Eye Muscle Shared Protein G2s: Autoantibodies against G2s Are Closely Associated with Ophthalmopathy in Patients with Graves’ Hyperthyroidism

2000 ◽  
Vol 85 (4) ◽  
pp. 1641-1647
Author(s):  
Kazuaki Gunji ◽  
Annamaria De Bellis ◽  
Audrey WU Li ◽  
Masayo Yamada ◽  
Sumihisa Kubota ◽  
...  
Keyword(s):  
Type 1 Diabetes ◽  
Immunosorbent Assay ◽  
Hashimoto’S Thyroiditis ◽  
Normal Subjects ◽  
Muscle Membrane ◽  
Hashimoto's Thyroiditis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Eye Muscle ◽  
Calculated Molecular Mass

Serum autoantibodies against eye muscle antigens are closely linked with thyroid-associated ophthalmopathy (TAO), although their significance is unclear. The two antigens that are most often recognized are eye muscle membrane proteins with molecular masses of 55 and 64 kDa, as determined from immunoblotting with crude human or porcine eye muscle membranes. We cloned a fragment of the 55-kDa protein by screening an eye muscle expression library with affinity-purified anti-55 kDa protein antibody prepared from a TAO patient’s serum. A complementary DNA (cDNA) encoding a novel protein, which we have called G2s, was sequenced on both strands, and its size was 411 bp. The open reading frame of G2s corresponded to a 121-amino acid peptide with a size of 1.4 kb. Using the rapid amplification of 5′-cDNA ends technique we were able to clone an additional 0.3 kb of the protein. G2s did not share significant homologies with any other entered protein in computer databases and had one putative transmembrane domain. Using the 1.4 kb cDNA as probe in Northern blotting of a panel of messenger ribonucleic acids prepared from human tissues, the parent protein was shown to correspond to a large molecule of about 5.8 kb with a calculated molecular mass of approximately 220 kDa, consistent with earlier immunoblot studies performed in the absence of reducing agents. G2s was strongly expressed in eye muscle, thyroid, and other skeletal muscle and to a lesser extent in pancreas, liver, lung, and heart muscle, but not in kidney or orbital fibroblasts. We tested sera from patients with Graves’ hyperthyroidism with and without ophthalmopathy and from control patients and subjects for antibodies against a G2s fusion protein by immunoblotting and enzyme-linked immunosorbent assay. In immunoblotting, antibodies reactive with G2s were identified in 70% of patients with TAO of less than 3 yr duration, 53% with TAO of more than 3 yr duration, 36% with Graves’ hyperthyroidism without evident ophthalmopathy, 17% with Hashimoto’s thyroiditis, 3% with type 1 diabetes, 23% with nonimmunological thyroid disorders, and 16% of normal subjects. The prevalences, compared to normal values, were significant for the two groups of patients with TAO, but not for the other groups. Tests were positive in 54% of patients with active TAO, 33% with chronic ophthalmopathy, 36% with Graves’ hyperthyroidism, 54% with Hashimoto’s thyroiditis, 23% with type 1 diabetes, and in 11% of normal subjects using enzyme-linked immunosorbent assay. The antibodies predicted the development of the ocular myopathy subtype of TAO in six of seven patients and the congestive ophthalmopathy subtype in seven of eight patients, respectively, with Graves’ hyperthyroidism studied prospectively during and after antithyroid drug therapy. Antibodies reactive with G2s may be early markers of ophthalmopathy in patients with Graves’ hyperthyroidism. Because G2s is expressed in both thyroid and eye muscle, immunoreactivity against a shared epitope in the two tissues may explain the well known link between thyroid autoimmunity and ophthalmopathy.

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.

Enzyme-linked immunosorbent assay of autoantibodies reacting with thyroid plasma membrane antigens in sera of patients with autoimmune thyroid diseases

1986 ◽  
Vol 113 (2) ◽  
pp. 255-260 ◽  
Author(s):  
Andrzej Gardas ◽  
Kathleen L. Rives
Keyword(s):  
Plasma Membrane ◽  
Immunosorbent Assay ◽  
Lupus Erythematosus ◽  
Antithyroid Drug ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Autoimmune Thyroid Diseases ◽  
Nodular Goitre ◽  
Membrane Antigens ◽  
Toxic Nodular Goitre

Abstract. A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of autoantibodies reacting with thyroid plasma membrane antigens has been established. Autoantibodies reacting with thyroid plasma membrane antigens were detected by the ELISA in 95% of untreated hyperthyroid Graves', 68% of antithyroid drug-treated Graves' up to four months of the therapy, in 62% of Hashimoto's thyroiditis and in 8.9% of toxic nodular goitre. The ELISA was negative in 100% healthy blood donors, 100% non-toxic nodular goitre, in 12 patients with rheumatoid arthritis, 18 patients with scleroderma and 94% of patients with systemic lupus erythematosus. The mean value of autoantibodies titre was higher in untreated hyperthyroid Graves' (1:84 000) and lowest in positive patients with autoimmune disease of non-thyroid origin (1:4000). The cross-reactivity of antimicrosomal antigen antibodies was below 10%; there was no influence of antithyroglobulin antibodies on the ELISA; and most of the autoantibodies react with plasma membrane antigens different from the TSH binding sites.

Lipoprotein(a) quantified by an enzyme-linked immunosorbent assay with monoclonal antibodies.

1989 ◽  
Vol 35 (7) ◽  
pp. 1380-1384 ◽  
Author(s):  
C Labeur ◽  
G Michiels ◽  
J Bury ◽  
D C Usher ◽  
M Rosseneu
Keyword(s):  
Monoclonal Antibody ◽  
Immunosorbent Assay ◽  
Plasma Cholesterol ◽  
Sample Pretreatment ◽  
Hdl Cholesterol ◽  
Normal Subjects ◽  
Enzyme Linked Immunosorbent Assay ◽  
Apo B ◽  
Lipoprotein A ◽  
Screening Programs

Abstract This new, sensitive, specific "sandwich"-type enzyme-linked immunosorbent assay (ELISA) for quantifying lipoprotein(a) [Lp(a)] in human serum and in ultracentrifugal lipoprotein fractions is based on use of a monoclonal antibody raised against apolipoprotein(a) as coating protein and a polyclonal antibody, raised against either apo B or against Lp(a) and conjugated with peroxidase, for detection of bound Lp(a). Mean intra- and interassay CVs for assay of 16 samples were 3.0% and 5.6%, respectively. Sample pretreatment with urea did not enhance Lp(a) immunoreactivity, and treatment with nonionic detergents decreased binding to the monoclonal antibody. Results correlated well (r = 0.99, n = 38) with those by radial immunodiffusion (RID). The ELISA assay, however, detects amounts corresponding to Lp(a) contents of 10 to 1000 mg/L in plasma samples diluted 1000-fold, compared with 100-500 mg/L for RID. For 92 normolipidemic subjects, the mean Lp(a) concentration was 120 (SD 130) mg/L. In patients undergoing coronary angiography, Lp(a) concentrations increased with the severity of the disease but were not correlated with either HDL cholesterol, triglycerides, apo A-I, or apo B, and only weakly with plasma cholesterol and apo A-II. These two correlations were even weaker in normal subjects, and only the correlation with total cholesterol was valid. Lp(a), measured at birth and at seven days and six months, steadily increased with age. This assay is well suited for measuring Lp(a) in plasma and in lipoprotein fractions and also for screening programs evaluating this significant genetic risk factor for the development of atherosclerosis.

scholarly journals Selection of single chain antibody fragments binding to the extracellular domain of 4-1BB receptor by phage display technology

Tumor Biology ◽  
2017 ◽  
Vol 39 (3) ◽  
pp. 101042831769592 ◽  
Author(s):  
Salman Bagheri ◽  
Mehdi Yousefi ◽  
Elmira Safaie Qamsari ◽  
Farhad Riazi-Rad ◽  
Mohsen Abolhassani ◽  
...  
Keyword(s):  
Phage Display ◽  
Immunosorbent Assay ◽  
Specific Binding ◽  
Interleukin 2 ◽  
Extracellular Domain ◽  
Surface Glycoprotein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Single Chain Variable Fragments ◽  
Selection Of

The 4-1BB is a surface glycoprotein that pertains to the tumor necrosis factor–receptor family. There is compelling evidence suggesting important roles for 4-1BB in the immune response, including cell activation and proliferation and also cytokine induction. Because of encouraging results of different agonistic monoclonal antibodies against 4-1BB in the treatment of cancer, infectious, and autoimmune diseases, 4-1BB has been suggested as an attractive target for immunotherapy. In this study, single chain variable fragment phage display libraries, Tomlinson I+J, were screened against specific synthetic oligopeptides (peptides I and II) designed from 4-1BB extracellular domain. Five rounds of panning led to selection of four 4-1BB specific single chain variable fragments (PI.12, PI.42, PII.16, and PII.29) which showed specific reaction to relevant peptides in phage enzyme-linked immunosorbent assay. The selected clones were successfully expressed in Escherichia coli Rosetta-gami 2, and their expression was confirmed by western blot analysis. Enzyme-linked immunosorbent assay experiments indicated that these antibodies were able to specifically recognize 4-1BB without any cross-reactivity with other antigens. Flow cytometry analysis demonstrated an acceptable specific binding of the single chain variable fragments to 4-1BB expressed on CCRF-CEM cells, while no binding was observed with an irrelevant antibody. Anti-4-1BB single chain variable fragments enhanced surface CD69 expression and interleukin-2 production in stimulated CCRF-CEM cells which confirmed the agonistic effect of the selected single chain variable fragments. The data from this study have provided a rationale for further experiments involving the biological functions of anti-4-1BB single chain variable fragments in future studies.

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