Background: Glutamic acid decarboxylase-65 (GAD65) is a major autoantigen in autoimmune diabetes and is discharged from injured islet beta cells. GAD65 may also be released by transplanted islets undergoing immunological rejection. To test hypotheses regarding the utility of GAD65 as a biomarker for transplant rejection or diabetes-associated islet damage and also regarding the timing and instigators of GAD65 release in humans or animal models, a sensitive assay capable of measuring GAD65 in serum or plasma will be necessary. Ideally, this assay would also be resistant to interference by anti-GAD65 autoantibodies.
Methods: A novel, magnetic bead-based assay was developed based on GAD65 capture by a monoclonal antibody directed to the only region of the protein known not to be significantly targeted by autoantibodies. A subsequent denaturation step allows sensitive immunodetection to proceed using anti-GAD65 polyclonal antibodies that would otherwise potentially be blocked by bound autoantibodies.
Results: The GAD65 assay worked equally well with serum and plasma as with a solution of bovine serum albumin (BSA). The limit of blank was 31 pg/mL and did not differ significantly in the BSA solution (27 pg/mL). Mean recovery of GAD65 from the plasma of control subjects and GAD65 autoantibody-positive and -negative subjects with type 1 diabetes was 101 +/- 4.6%, 88 +/- 7.8%, and 99 +/- 7.0% (+/- SEM), respectively. The assay was used to quantify both recombinant GAD65 and the GAD65 content of human and rodent islets and other tissue extracts that were added to human plasma samples.
Conclusions: A sensitive, autoantibody-resistant GAD65 assay has been developed that is compatible with detection in serum and plasma and therefore will likely also work with a variety of other biologic fluids. This assay may enable the use of circulating GAD65 as a biomarker of islet damage or transplant rejection and will facilitate in vivo studies of the pathogenesis of anti-GAD65 autoreactivity.