Identifying autoantibody biomarkers for Addison’s disease in dogs

Abstract

Abstract Addison’s disease is a life-threatening endocrinopathy caused by the immune-mediated destruction of the adrenal cortex. Only dogs and humans develop the disease naturally. In humans, autoantibodies against the adrenocortical steroidogenic enzyme 21-hydroxylase can predict patients at high risk of developing the disease. No comparable biomarker exists for canine Addison’s disease. We hypothesized that, similar to humans, newly diagnosed dogs with Addison’s disease will have consistently detectable autoantibodies against adrenocortical steroidogenic enzymes. Serum was collected from dogs in four study cohorts (diagnosed within 30 days and healthy age-matched controls; long-term cases and healthy age-matched controls) in three dog breeds with a high prevalence of Addison’s disease. These samples were evaluated for anti-adrenocortical autoantibodies using both immunofluorescence assays and Western blotting. Initial results from immunofluorescence assays on bovine adrenal sections probed with serum IgGs from the recently diagnosed cohort showed positive staining in the adrenal cortex and negative staining for the unaffected cohorts. Additionally, preliminary data from Western blots on canine adrenal homogenates probed with serum IgGs identified a band at ~42 kD unique to the recently diagnosed group from each breed. These findings have been replicated using five canine adrenal homogenates, as well as bovine and porcine homogenates. Our results suggest that dogs with a new diagnosis of Addison’s disease may have autoantibodies targeting a ~42 kD adrenal protein. Ongoing work includes testing reproducibility and identifying the specific antigenic target using 2D gels with spot cutting followed by LC-MS/MS.