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 th yroid (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-polyac rylamide 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 exam ined histologically at sacrifice, Following immunization serum and, in some cases, the corresponding MCAB produced by fusion of the mouse sp leen cells with a mouse myeloma cell line, were tested for EM and THY cross-reactivity in an ELISA and by immunoblotting. The number of anti bodies demonstrated in Western blotting identified as bands of reactiv ity, and ELISA levels, expressed as optical density - increased with t ime, each peaking at around 10-12 weeks. THY and EM antibody cross-rea ctivity was demonstrated in the majority of mice, serum from mice immu nized with THY membranes reacting with these membranes as well as with pig EM membranes in both ELISA and immunoblotting and, conversely, se ra from mice immunized with pig EM membranes also reacting with THY me mbranes 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-ass ociated ophthalmopathy. There was also some cross-reactivity with brai n membranes, used as control antigen in both tests and in immunization , although to a lesser degree,, but very little to liver and orbital c onnective tissue membrane. While there were several exceptions there w as, 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 no ne developed features of an ophthalmopathy although the experimental p rotocol was not optimised for the production of autoimmunity. This is the first report of the production of antibodies reactive against puta tive THY and EM shared epitopes in experimental animals and provides f urther support for the notion that the mechanism for the;association o f ophthalmopathy with autoimmune THY disorders in humans may be immuno logical crossreactivity.