Experiences with the SELDI-TOF Method for the Detection of the Diabetes Associated Antigen GAD 65 in the Peripheral Blood of Patients with Diabetes and Inflammation

Abstract

Abstract The treatment of diabetes mellitus type 1 is a major challenge in health care. The diabetes associated antigen Glutamic Acid Decarboxylase (GAD 65) has been described as strictly intracellular. However an intracellular antigen would never have access to the immune system. In a previous study with the high sensitive fluorescence correlation spectroscopy (FCS), we detected the GAD 65 antigen in the sera of patients with type 1 diabetes and related autoimmune diseases. FCS is a time consuming measurement procedure and very sensitive to disturbance, which makes unsuitable for a routine screening of the patient sera for GAD 65. Since the measured GAD 65 concentrations were surprisingly high (2.65 microgram/ml) and therefore within the detection range of less sensitive methods, in this study we used the SELDI-TOF (Surface enhanced laser desorption ionisation - time of flight) technique to confirm the FCS results in view of an early diabetes diagnosis. For this purpose we developed an array with the SELDI-TOF method and analysed 4 patient sera (2 diabetes and 2 controls) in a pilot experiment. For one diabetes serum, we found some evidence for the diabetic antigen GAD 65 in the mass spectra in a peak close to albumin with a molecular weight of 65 kDa. The 3 other samples remained negative. Because of the higher concentration of albumin in the sera, a peak evaluation in regard of GAD 65 was difficult and not reliable in this setup. Troubles and experiences with SELDI-TOF for the detection of GAD 65 are discussed.