Time-resolved Fluorometric Assay for Detection of Autoantibodies to Glutamic Acid Decarboxylase (GAD65)

Abstract

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

Murine High Specificity/Sensitivity Competitive Europium Insulin Autoantibody Assay

“…10 No standard ELISA has performed as well as their respective radioassays in international workshops of the IDS and Diabetes Autoimmunity Standardization Program (DASP), while a modified assay utilizing single chains of the antibody-one to bind to GAD on the well and the other to biotinylated GAD in solution-performed very well in the last DASP workshop. We have recently described a europium competitive assay for autoantibodies reacting with interferon-a that specifically detects autoantibodies present in patients with the autoimmune polyendocrine syndrome type I.…”

“…Therefore, europium has been utilized in ELISAformat assays to provide high sensitivity, comparable to radioassays, with time-resolved fluorescent detection of photons (results reported as counts per second [cps]). 10,11 Some control sera we analyzed, however, gave false-positives (greater signal with insulin on the plate than without platebound insulin), and we further modified the europium assay to incorporate competition with excess fluid-phase insulin, with results, and thus positivity, determined by the difference with and without competition. The final competitive europium insulin autoantibody assay (CE-IAA) analyzing sera from mice is superior in sensitivity to our standard mIAA assay, and much simpler, requiring relatively inexpensive reagents and 1-s counting and not requiring radioactive compounds or chemically modified autoantigens.…”

Evaluation of Two Nonisotopic Immunoassays for Determination of Glutamic Acid Decarboxylase and Tyrosine Phosphatase Autoantibodies in Serum

“…Autoantibodies against ␤-cells can be detected before clinical onset of DM1 (2,3 ) and can help to distinguish type 2 diabetes mellitus (DM2) from latent autoimmune diabetes of the adult (LADA) (4 ), which may represent one-half of cases of autoimmune diabetes (1 ). The major autoantibodies in DM1 are against islet cells, insulin, a protein tyrosine phosphatase-like protein (IA-2), and the 65-kDa isoform of glutamic acid decarboxylase (GAD65) (1 ).…”

“…RBAs, although cumbersome and requiring use of 35 S, reportedly provide more precise results and higher diagnostic sensitivity and specificity than ELISAs (2)(3)(4).…”

Time-Resolved Immunofluorometric Dual-Label Assay for Simultaneous Detection of Autoantibodies to GAD65 and IA-2 in Children with Type 1 Diabetes

“…We previously published single-label time-resolved immunofluorometric assay (TR-IFMA) methods for the detection of GADAs and IA-2As in a solid-phase streptavidin-coated 96-well microtiter plate format (12,13 ). The detection uses europium lanthanide chelate as a fluorescent label.…”