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

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 \m=x\g pellet, a pellet depleted of the 100 000 \ m=x\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'

Smoking and Immunity: an Additional Player in the Mosaic of Autoimmunity

“…The component towards which antibodies were found to bind has been characterized as a 64 kDa extraocular muscle protein [45].…”

The Ocular Muscle Cell Is a Target of the Immune System in Endocrine Ophthalmopathy

“…Contra dictory data reported by another group who used expanded orbital lymphocytes [49] could be explained by a modifica tion of T cell function during tissue culture [48], Results o f the many studies on humoral immunity in TAO have been summarized recently by Nauman [50] and Wall [51], Since the first report of circulating antibodies re active with a soluble (cytosolic) EM antigen [52] consid erable evidence has been obtained to support the EM as the primary target of autoantibodies in TAO. Serum EM-rcactive antibodies have been detected using ELISA [53][54][55][56], Staphylococcus protein A binding [57,58], immunoblotting [59][60][61], antibody-dependent cell-mediated cytotoxicity (ADCC) [62. 63] and immunofluorescence [64][65][66].…”

“…Since the concentrations of these tissue fractions required to adsorb EM antibodies were generally greater than for EM Hum oral Im m unity against E M A n tig e n s [50], the former appears more likely. ELISA, although sim ple and convenient to perform, was found to have limited values in studies ofTAO when crude preparations were used as antigen [54,56], Despite these technical difficulties, the existence of serum EM antibodies in TAO has been well es tablished. and the significant correlations of their levels with parameters o f the eye disorder suggest that they may have a pathogenic role [71,72].…”

The Fibroblast Is the Target Cell in the Connective Tissue Manifestations of Graves' Disease

“…Patients with GO have circulating antibodies that are di rected against eye muscle and orbital connective tissue [2,28]. A 64-kD cxtraocular muscle protein and a 23-kD orbi tal fibroblast protein have been identified as antigens against which these antibodies are directed [6,29], Howev er, these proteins are not restricted to orbital tissue, and the antibodies are present in normal individuals [3-5, 8, 9, 29], suggesting that these antibodies are not causative in the dis ease.…”

“…Due primarily to difficulties inherent in culturing muscle cells, and to the lack of available human eye tissue, early studies generally used crude preparations of soluble nonhuman eye muscle antigen. Although these early studies reported the presence of circulating antibodies directed against eye muscle antigen [1,2], the findings were not con firmed by other investigators [3] or the antibodies were found to be nonspecific [4,5], Later studies concerning a 64-kD eye muscle membrane antigen were more convinc ing [6,7], However, it is difficult to implicate this protein as a target in the disease as it is present in a wide variety of tissues not involved in Graves' disease [8], reactivity against this antigen is demonstrable in normal serum [9], and hist ologic examination of affected tissues in active GO shows the muscle cells to be intact [10][11][12],…”