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1 Department of Biochemistry and Pharmacy, Åbo Akademi University, FIN-20521 Turku, Finland.
2 Department of Virology, University of Turku, FIN-20520 Turku, Finland.
3 Hospital for Children and Adolescents, University of Helsinki, FIN-00029 HUS, Finland.
4 Department of Pediatrics, Tampere University Hospital, FIN-33520 Tampere, Finland.
5 Department of Pediatrics, University of Oulu, FIN-90014 Oulu, Finland.
6 PerkinElmer Life and Analytical Sciences, Wallac Oy, FIN-20101 Turku, Finland.
aAddress correspondence to this author at: Department of Biochemistry and Pharmacy, Åbo Akademi University, PO Box 66, FIN-20521 Turku, Finland. Fax 358-2-215-4745; e-mail mankelo{at}abo.fi.
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 4 140 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.
The following articles in journals at HighWire Press have cited this article:
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  M. Ankelo, A. Westerlund, K. Blomberg, M. Knip, J. Ilonen, and A. E. Hinkkanen Time-Resolved Immunofluorometric Dual-Label Assay for Simultaneous Detection of Autoantibodies to GAD65 and IA-2 in Children with Type 1 Diabetes Clin. Chem., March 1, 2007; 53(3): 472 - 479. [Abstract] [Full Text] [PDF]
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X. Palomer, D. Mauricio, J. Rodriguez-Espinosa, E. Zapico, C. Mayoral, F. Gonzalez-Sastre, A. de Leiva, and F. Blanco-Vaca Evaluation of Two Nonisotopic Immunoassays for Determination of Glutamic Acid Decarboxylase and Tyrosine Phosphatase Autoantibodies in Serum Clin. Chem., August 1, 2004; 50(8): 1378 - 1382. [Abstract] [Full Text] [PDF]
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