Cross-reactive antibodies in the serum of balb/c mice immunized with thyroid or eye muscle membranes.

During the course of immunizing balb/c mice with eye muscle (EM) or thyroid (THY) membranes for monoclonal antibody (MCAB) production their sera frequently contain antibodies which react against both EM and THY membranes in enzyme-linked immunosorbent assay (ELISA) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. In order to further study this phenomenon we have analyzed sera from 27 balb/c mice, including 10 that were studied serially, and their tissues examined histologically at sacrifice. Following immunization serum and, in some cases, the corresponding MCAB produced by fusion of the mouse spleen cells with a mouse myeloma cell line, were tested for EM and THY cross-reactivity in an ELISA and by immunoblotting. The number of antibodies demonstrated in Western blotting identified as bands of reactivity, and ELISA levels, expressed as optical density--increased with time, each peaking at around 10-12 weeks. THY and EM antibody cross-reactivity was demonstrated in the majority of mice, serum from mice immunized with THY membranes reacting with these membranes as well as with pig EM membranes in both ELISA and immunoblotting and, conversely, sera from mice immunized with pig EM membranes also reacting with THY membranes in the two tests. In Western blotting a variety of THY and EM-reactive antibodies were demonstrated including those directed against a 64 kDa protein, shown to be an important autoantigen in thyroid-associated ophthalmopathy. There was also some cross-reactivity with brain membranes, used as control antigen in both tests and in immunization, although to a lesser degree, but very little to liver and orbital connective tissue membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies reactive with an intracellular epitope of a recombinant 64 kDa thyroid and eye muscle protein in patients with thyroid autoimmunity and ophthalmopathy.

We have developed an enzyme-linked immunosorbent assay (ELISA) for the measurement of antibodies reactive with a 98 amino acid fragment, called D1, of a recombinant thyroid and eye muscle membrane protein corresponding to a MW of 64 kDa (called 1D) in the serum of patients with thyroid autoimmunity with and without ophthalmopathy. Antibodies against the D1 fragment expressed as a fusion protein with beta galactosidase, were detected in 29% of patients with thyroid-associated ophthalmopathy (TAO) of < 1 yr duration, in 33% of those with disease of > 3 yr duration, in 40% of patients with Graves' hyperthyroidism (GH) without evident eye disease, in 31% of patients with lid lag and retraction but no other signs of progressive ophthalmopathy, in 25% of patients with euthyroid Graves' disease and in 43% of patients with untreated Hashimoto's thyroiditis (HT), but in none of 14 patients with other (non-immunological) thyroid disorders. Although tests were positive in 6 out of the 15 patients with ophthalmopathy and no overt thyroid autoimmunity overall there was no close association of the antibodies with clinical features of the eye disease or its course. In those sera in which Western blotting for antibodies reactive with a 64 kDa eye muscle membrane protein and ELISA were both carried out there was no close correlation between the two tests.(ABSTRACT TRUNCATED AT 250 WORDS)

Nature and significance of orbital autoantigens and their corresponding autoantibodies in thyroid-associated ophthalmopathy.

There is now considerable evidence that the pathogenesis of thyroid-associated ophthalmopathy is closely linked to the presence of a shared autoantigen(s) in the thyroid and the eye muscle, against which cytotoxic mechanisms are directed. Although the orbital connective tissue is certainly involved in the orbital inflammatory process, a 64 kDa membrane protein expressed by both the eye muscle and the thyroid and recognized consistently by antibodies in the sera of TAO patients, seems to be the most likely target candidate. While its presence in non ocular skeletal muscle is not as well established, more recent data tend to suggest the existence of a 64 kDa molecule in the three tissues. The availability of a cDNA encoding a 572 amino acid protein corresponding to a MW of 63-64 kDa, which may be the same molecule, will allow us to determine more clearly the structural characteristics of the different molecules proposed as targets. The role of the corresponding autoantibodies in the pathogenesis of the eye disease is far less well defined. Whether they play a role in the induction of the ophthalmopathy or only represent helpful markers remains to be clarified.

Muscle and species reactivity of mouse monoclonal antibodies to human eye muscle membrane antigens.

We studied the tissue and species reactivity of mouse monoclonal antibodies (MCAB) produced by immunizing mice with a 100,000g ultracentrifuged preparation of human eye muscle (HEM) membranes. Twenty-three MCABs, 20 of which reacted in an enzyme-linked immunosorbent assay (ELISA) with HEM membrane, 2 with human thyroid membrane, and 1 nonreactive negative control, were selected for the study. The muscle and species specificity of 6 of the most reactive and more restrictively reactive MCAB were studied in more detail. All reacted in ELISA with human skeletal muscle membrane and, to a lesser extent, with human cardiac muscle membrane, but not with human brain membrane. The 6 MCAB cross-reacted with eye muscle membrane prepared from pig but not rat, although reactivity with human tissue was greatest for all MCAB tested. When tested in immunoblotting with HEM and thyroid membranes, 3 of 6 MCAB reacted with a 64-kDa protein in HEM, 2 of which also reacted with an antigen of the same molecular weight in thyroid membrane. In a complement-mediated antibody-dependent cytotoxicity assay, 5 of 19 MCAB lysed HEM cells, 6 of 21 lysed human skeletal muscle cells, and 10 of 22 lysed human thyroid cells. These findings support results from earlier clinical studies which showed that eye muscle membrane reactive autoantibodies in the serum of patients with thyroid-associated ophthalmopathy cross-react with membrane prepared from other striated muscle. The significance of eye muscle, skeletal muscle, and thyroid cross-reactivity of MCAB is discussed in the context of autoimmune thyroid disease and ophthalmopathy.

Eye muscle membrane reactive antibodies are not detected in the serum or immunoglobulin fraction of patients with thyroid-associated ophthalmopathy using an ELISA and crude membranes.

We tested sera and purified immunoglobulin (Ig) fractions from patients with autoimmune thyroid disorders (AITD), with and without ophthalmopathy, and normal subjects, for the presence of antibodies reactive with eye muscle membrane antigens in an optimized enzyme-linked immunosorbent assay (ELISA). We found no correlation between ELISA results and the presence or severity of ophthalmopathy in patients with AITD for either serum or Ig, and there were no significant differences between the mean values (+/- SE) for the three groups (AITD with ophthalmopathy, AITD without ophthalmopathy and normals) for either serum or Ig. In contrast Ig from 8 of 19 (45%) patients with thyroid-associated ophthalmopathy reacted with a 64 kDa eye muscle membrane antigen in SDS-polyacrylamide gel electrophoresis and Western blotting, while tests were positive in only one of the 8 patients with AITD without eye disease and in none of the 8 normal subjects. The presence of antibodies to a 64 kDa antigen in immunoblotting did not correlate with the levels of antibodies measured in ELISA. We conclude that the ELISA, incorporating a crude membrane fractions as antigen, is not useful as a clinical test for eye muscle autoantibodies.

Direct leukocyte migration inhibition by myelin basic protein in exacerbations of multiple sclerosis.

Studies in 13 normal subjects, 9 patients with multiple sclerosis (MS) within 3 weeks of exacerbation and 16 others 1 to 6 months after onset were carried out for evidence of cell-mediated hypersensitivity to myelin basic protein. Ten patients with stroke and 10 with Guillain-Barré syndrome were studied as additional controls. Peripheral leukocytes obtained by leukapheresis were packed into capillary tubes and allowed to migrate out onto glass in the presence or absence of myelin basic protein. Cells of patients within 3 weeks of an MS episode gave a mean migration index of 68 +/- 9%, and those 1 to 6 months after onset, 93 +/- 21%. For the entire MS group the mean index was 88 +/- 20%, for those with Guillain-Barré, 103 +/- 7%; and for the stroke patients, 107 +/- 11%. Results for the acutely ill MS patients were significant (P less than 0.005). The data are similar to those obtained using the migration inhibition factor assay but show that sensitized lymphocytes also elaborate a second mediator during acute exacerbations of illness. These observations strengthen evidence that sensitization to this potent encephalitogen occurs simultaneously with exacerbations of clinical illness.