Determination And Applications Of Glutamic Acid Decarboxylase (GAD₆₅) Reactive T-cells In Diabetes

Part 1Development of enzyme-linked immunospot (ELISPOT) assay for GAD₆₅-reactive T cells in diabetesObjective: To develop an optimized ELISPOT assay for detection of GAD₆₅-autoreactive T lymphocytes(ART).Methods: Two patients with classical type 1 diabetes(T1A), 2 with idiopathic type 1 diabetes(T1B) , 3 with type 2 diabetes(T2DM), and 2 with healthy controls were investigated. Human peripheral blood mononuclear cells(PBMC) were isolated by Ficoll-Hypaque density gradients. Freezing and thawing of human PBMC were performed correctly according to the protocol of ELISPOT of U-CyTech Company(Dutch). Different incubation methods, incubation periods, stimulation antigen, cells concentration and cryopreservation of PBMC or not were observed to find an optimized spot-forming way. Reproducibility of ELISPOT assay was also assessed.Results: â‘ No significant differences on IFN-γ-spots and IL-4 spots between assay without preincubation and with preincubation were found, whereas assay without preincubation was easier to perform. â‘¡The optimal antigen concentration and incubation time were stimulation of 5...