Detection of GAD65-reactive T-Cells in type 1 diabetes by immunoglobulin-free ELISPOT assays.
OBJECTIVE
To investigate the prevalence of beta-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.


METHODS
Cellular immune responses to beta -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 gamma-interferon (IFN-gamma)-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-gamma 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 beta-cell autoantibody determination, will improve the diagnosis of type 1 diabetes.
Kotani
Reiko
R
Division of Internal and Geriatric Medicine, Department of Development and Aging, Kobe University Graduate School of Medicine, Kobe, Japan.
Nagata
Masao
M
Moriyama
Hiroaki
H
Nakayama
Maki
M
Yamada
Katsumi
K
Chowdhury
Shahead Ali
SA
Chakrabarty
Sagarika
S
Jin
Zhenzi
Z
Yasuda
Hisafumi
H
Yokono
Koichi
K
D000293:Adolescent; D000328:Adult; D000368:Aged; D001323:Autoantibodies; D001324:Autoantigens / Q000032:analysis / Q000276:immunology; D002364:Caseins / Q000032:analysis / Q000276:immunology; D002455:Cell Division / Q000276:immunology; D003922:Diabetes Mellitus, Type 1 / Q000175:diagnosis* / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D005968:Glutamate Decarboxylase / Q000032:analysis* / Q000276:immunology; D006801:Humans; D007515:Islets of Langerhans / Q000276:immunology; D007527:Isoenzymes / Q000032:analysis* / Q000276:immunology; D008297:Male; D008875:Middle Aged; D013601:T-Lymphocytes / Q000166:cytology / Q000201:enzymology*
D001323:Autoantibodies; D001324:Autoantigens; D002364:Caseins; D007527:Isoenzymes; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2