A highly sensitive immunoassay resistant to autoantibody interference for detection of the diabetes-associated autoantigen glutamic acid decarboxylase 65 in blood and other biological samples.

Background: Glutamic acid decarboxylase-65 (GAD65) is a major autoantigen in autoimmune diabetes and is discharged from injured islet β 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: Th...