Analysis of Autoantibody Epitopes on Steroid 21-Hydroxylase Using a Panel of Monoclonal Antibodies

A panel of five mouse monoclonal antibodies (MAbs) to human recombinant steroid 21-hydroxylase (21-OH) were produced, characterized, and used to study the interaction of 21-OH autoantibodies (AAbs) with different epitopes on human 21-OH. AAbs in patients with isolated autoimmune Addison’s disease, autoimmune polyglandular syndromes types I and II, and 21-OH antibody-positive patients without overt Addison’s disease (25 patients in total) were studied. Four MAbs were IgG1 subclass, one was IgG2a, and all had κ light chains. The affinities of four of the antibodies were in the range 2.0 × 108 m−1 to 7.0 × 108 m−1, and the affinity of the other was 2.3 × 107 m−1. 21-OH MAbs did not cross-react with 17α-hydroxylase (17α-OH) or P450 side chain cleavage enzyme. Studies using a series of 21-OH fragments allowed the identification of short stretches of amino acids (AA) that were involved in forming the MAb binding sites. AA 391–405, defined as epitope region (ER) 1, were found to be important for binding of M21-O...