Detection of GAD65-Reactive T-Cells in Type 1 Diabetes by Immunoglobulin-Free ELISPOT Assays
- Reiko Kotani, MD,
- Masao Nagata, MD, PHD,
- Hiroaki Moriyama, MD, PHD,
- Maki Nakayama, MD,
- Katsumi Yamada, MD,
- Shahead Ali Chowdhury, MD,
- Sagarika Chakrabarty, MD,
- Zhenzi Jin, MD,
- Hisafumi Yasuda, MD, PHD and
- Koichi Yokono, MD, PHD
- Division of Internal and Geriatric Medicine, Department of Development and Aging, Kobe University Graduate School of Medicine, Kobe, Japan
Abstract
OBJECTIVE—To investigate the prevalence of β-cell autoantigen-reactive peripheral T-cells in type 1 diabetes, we developed an immunoglobulin-free enzyme-linked immunospot (ELISPOT) assay and assessed its usefulness for diagnosing this disease.
RESEARCH DESIGN AND METHODS—Cellular immune responses to β -cell autoantigens were studied both by immunoglobulin-free proliferation assays and ELISPOT assays in 33 patients with type 1 diabetes and 15 patients with type 2 diabetes, compared with 23 healthy control subjects. Autoantibodies against GAD65 and IA-2 were measured by radioimmunoassay.
RESULTS—Significant proliferative responses to GAD65 were observed in 10 of 31 (32.3%) type 1 diabetic patients (P < 0.05), whereas GAD65-reactive γ-interferon (IFN-γ)-secreting cells were detected in 22 of 33 patients (66.7%) by ELISPOT assay (P < 0.001). Of patients negative for both GAD65 and IA-2, five of six (83.3%) showed IFN-γ positivity in ELISPOT and two of five (40.0%) showed significant proliferation against GAD65.
CONCLUSIONS—Using a newly developed ELISPOT assay, GAD-reactive T-helper 1 cells in PBMC of type 1 diabetic patients could be identified at a higher frequency than by the proliferation assay. Therefore, the immunoglobulin-free ELISPOT assay is an excellent tool for detecting T-cell reactivity to autoantigens with greater specificity and, in combination with β-cell autoantibody determination, will improve the diagnosis of type 1 diabetes.
- ELISPOT, enzyme-linked immunospot
- IFN-γ γ-interferon
- IL-4, interleukin-4
- mAb, monoclonal antibody
- PBMC, peripheral blood mononuclear cell
- PHA, phytohemagglutinin
- SI, stimulation index
- Th1, T-helper 1
- MW, molecular weight
Footnotes
-
Address correspondence and reprint requests to Masao Nagata, MD, PhD, Division of Internal and Geriatric Medicine, Department of Development and Aging, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail: nagata{at}med.kobe-u.ac.jp.
Received for publication 22 November 2001 and accepted in revised form 23 April 2002.
A table elsewhere in this issue shows conventional and Système International (SI) units and conversion factors for many substances.
- DIABETES CARE
