Detection of autoantibodies to protein tyrosine phosphatase-like protein IA-2 with a novel time-resolved fluorimetric assay.
BACKGROUND
Circulating autoantibodies to pancreatic glutamic acid decarboxylase (GAD65; the 65-kDa isoform of glutamic acid decarboxylase), protein tyrosine phosphatase-like protein IA-2, and insulin can be used as predictive markers of type 1 diabetes. We developed a novel assay for the detection of IA-2 autoantibodies (IA-2As) in serum based on time-resolved fluorimetry, hypothesizing that this kind of assay could provide several advantages over methods described to date, including radiobinding assays (RBAs) and ELISAs.


METHODS
The intracellular part of IA-2 (IA-2ic) was biotinylated and bound to streptavidin-coated 96-well plates by simultaneous incubation with serum samples and glutathione S-transferase (GST)-IA-2ic fusion protein. GST-IA-2ic captured by autoantibodies in the serum was detected with europium-labeled anti-GST antibody, and the signal was measured in a time-resolved fluorimeter. A serum sample panel from 100 patients with newly diagnosed type 1 diabetes and 100 unaffected controls was analyzed with the new assay and a conventional RBA.


RESULTS
Among the 100 serum samples from patients with type 1 diabetes, the time-resolved fluorimetric assay identified 74 IA-2A-containing sera, whereas the RBA detected 80 IA-2A-positive samples. Five of the six samples positive in the RBA but not detected by the time-resolved fluorimetric assay were only weakly positive in the RBA. The performance time of the time-resolved fluorimetric assay was 2.5 h compared with 10-12 h required by the RBA.


CONCLUSIONS
The time-resolved fluorimetric assay provides a simple, nonradioactive analysis method for the detection of IA-2As with a specificity and a sensitivity comparable to the RBA method. This assay allows substantial reduction in performance time compared with the conventional RBA.
Westerlund-Karlsson
Annette
A
Department of Biochemistry and Pharmacy, Abo Akademi University, FIN-20521 Turku, Finland. awesterl@abo.fi
Suonpää
Katriina
K
Ankelo
Matti
M
Ilonen
Jorma
J
Knip
Mikael
M
Hinkkanen
Ari E
AE
D000293:Adolescent; D001323:Autoantibodies / Q000097:blood*; D001324:Autoantigens; D015415:Biomarkers / Q000097:blood; D002614:Chelating Agents; D002648:Child; D003922:Diabetes Mellitus, Type 1 / Q000097:blood / Q000175:diagnosis*; D005063:Europium; D015200:Fluoroimmunoassay; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology*; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000276:immunology*; D011869:Radioligand Assay; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001323:Autoantibodies; D001324:Autoantigens; D015415:Biomarkers; D002614:Chelating Agents; C096322:ICA512 autoantibody; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D005063:Europium; D005982:Glutathione Transferase; C516862:PTPRN protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8