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

Abstract

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 enzymelinked 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.