Time-resolved fluorometric assay for detection of autoantibodies to glutamic acid decarboxylase (GAD65).
BACKGROUND
Type 1 diabetes mellitus results from destruction of the pancreatic insulin-producing beta cells by a chronic autoimmune process. Methods are needed for the detection of circulating autoantibodies to glutamic acid decarboxylase (GAD65), a major marker of this process.


METHODS
Streptavidin-coated microtiter plates were incubated with biotinylated GAD65, and after incubation with serum samples from patients with type 1 diabetes mellitus and control individuals, europium-labeled GAD65 was added. After washing steps, the delayed fluorescence was measured in duplicate in a fluorometer. Samples collected from 100 patients with newly diagnosed type 1 diabetes mellitus and 100 healthy controls were measured by the new assay and by a radiobinding assay.


RESULTS
The detection limit of the new assay was 1.49 WHO units/mL, the calibration curve was linear to 4140 WHO units/mL, and no hook effect was observed up to 41,400 WHO units/mL. The intraassay CV was 2.1-6.3% over the calibration range. For patient serum samples, the intraassay, interassay, and total CVs were 5.4-7.0%, 9.8-13%, and 12-14%, respectively. Compared with conventional radioimmunologic methods, the analytical range was broader and the analysis time required to perform the measurements was shorter. At a cutoff with 99% specificity, the new assay and the radiobinding assay were positive in 71 and 67 patients, respectively.


CONCLUSIONS
The new assay provides a rapid and sensitive nonradioactive method applicable for large-scale screening for beta-cell autoimmunity. It has a broad linear analytical range, is easy to perform and automate, and has sensitivity and specificity comparable to those for the conventional radioisotope assay.
Ankelo
Matti
M
Department of Biochemistry and Pharmacy, Abo Akademi University, FIN-20521 Turku, Finland. mankelo@abo.fi
Westerlund-Karlsson
Annette
A
Ilonen
Jorma
J
Knip
Mikael
M
Savola
Kaisa
K
Kankaanpää
Pasi
P
Meriö
Liisa
L
Siitari
Harri
H
Hinkkanen
Ari
A
D000293:Adolescent; D001323:Autoantibodies / Q000097:blood*; D015415:Biomarkers / Q000097:blood; D002614:Chelating Agents; D002648:Child; D003922:Diabetes Mellitus, Type 1 / Q000175:diagnosis*; D005063:Europium; D015200:Fluoroimmunoassay; D005968:Glutamate Decarboxylase / Q000276:immunology*; D006801:Humans; D008297:Male; D011869:Radioligand Assay; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001323:Autoantibodies; D015415:Biomarkers; D002614:Chelating Agents; D005063:Europium; D005968:Glutamate Decarboxylase