Detection of autoantibodies to the diabetes-associated antigen IA-2 by a sensitive enzyme-linked immunosorbent assay.
The tyrosine phosphatase like protein IA-2 is an important autoantigen in insulin-dependent diabetes mellitus (type 1 diabetes). Autoantibodies to IA-2 (IA-2A) are present in the serum of patients with type 1 diabetes even before the onset of the disease. Previously, we reported on a radioimmune assay to detect IA-2A, using E. coli-derived 125I-labelled IA-2 as antigen. Although this assay could be shown to be equivalent to the common reference method for IA-2A detection (radioligand assays using in vitro synthesised 35S-methionine labelled antigen), the disadvantages of both assays with respect to synthesis and handling of the radioactive antigen limit their use in routine laboratories. In this study, we have evaluated a non-radioactive enzyme-linked immunosorbent assay (ELISA) for the simple detection of IA-2A. We report on an ELISA where the biotinylated intracytoplasmic part of IA-2 (IA-2ic) is captured on streptavidin-coated plates. The sensitivity of the ELISA was similar to the validated radioligand assay, as it detected 47 of 69 (69%) patients with type 1 diabetes as compared to 46 of 68 (67 %) with the reference method for IA-2A detection (radioligand assays using in vitro synthesised 35S-methionine labelled antigen). Only 2 of 50 (4%) patients with autoimmune thyroid disease and 1 of 114 (1 %) healthy controls were detected in the ELISA, confirming specificity. There was a significant correlation between the ELISA and the radioligand assay (r = 0.64, p<0.001). We conclude that this ELISA is suitable to detect IA-2A in the serum of patients with type 1 diabetes with a similar sensitivity and specificity to the radioligand assay. This ELISA will allow rapid and simple measurement of IA-2A where the radioligand assay is inconvenient or not available.
Löbner
K
K
Department of Internal Medicine III, University of Leipzig, Germany.
Khoo-Morgenthaler
U Y
UY
Seissler
J
J
Morgenthaler
N G
NG
Scherbaum
W A
WA
D000918:Antibody Specificity; D001323:Autoantibodies / Q000097:blood* / Q000302:isolation & purification; D001324:Autoantigens; D015153:Blotting, Western; D003922:Diabetes Mellitus, Type 1 / Q000201:enzymology / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins / Q000276:immunology*; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000276:immunology*; D011869:Radioligand Assay; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012680:Sensitivity and Specificity; D019809:Streptavidin; D013462:Sulfur Radioisotopes
D001323:Autoantibodies; D001324:Autoantigens; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D013462:Sulfur Radioisotopes; D019809:Streptavidin; C516862:PTPRN protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8