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

Abstract

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. While there were several exceptions there was, overall, a fairly close correlation between serum reactivities and tissue specificities of the corresponding MCAB. No mouse demonstrated significant lymphocytic infiltration in THY or orbital tissues and none developed features of an ophthalmopathy although the experimental protocol was not optimised for the production of autoimmunity. This is the first report of the production of antibodies reactive against putative THY and EM shared epitopes in experimental animals and provides further support for the notion that the mechanism for the association of ophthalmopathy with autoimmune THY disorders in humans may be immunological cross-reactivity.