Detection of Autoantibodies to the Diabetes-Associated Antigen IA-2 by a Sensitive Enzyme-Linked Immunosorbent Assay

Abstract

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.

Detection of Autoantibodies to Protein Tyrosine Phosphatase-like Protein IA-2 with a Novel Time-resolved Fluorimetric Assay

“…Other methods used are RIAs involving 125 Ilabeled antigens (10,19 ) and ELISAs (11,20 ). These assays need complex synthesis of reagents or require a long performance time, and they frequently require the handling of radioactive substances.…”

“…In that study, the recombinant IA-2ic was labeled with 125 I and was used in a RIA that gave a good correlation (r ϭ 0.79) with the standard RBA. In another study by the same authors, the biotinylated IA-2ic fusion protein was applied in an ELISA with a somewhat lower correlation with the RBA (r ϭ 0.64) (11 ). We expressed IA-2ic as a GST fusion protein that could be easily purified by affinity chromatography on a Glutathione Sepharose column.…”

“…In addition, a RIA based on the use of 125 I-labeled IA-2ic and immunoprecipitation has been developed (10 ). Other methods include ELISAs, in which the immobilized antigen captures antibodies from the sample and detection is achieved with labeled anti-human IgG antibody (11 ).…”

A Rapid Lateral Flow Immunoassay for the Detection of Tyrosine Phosphatase-Like Protein IA-2 Autoantibodies in Human Serum

“…In addition, they have the disadvantage of requiring special precautions and licensing because radioactive isotopes are used. Other methods for the detection of IA-2As use enzyme-linked immunosorbent assays (ELISAs) and time-resolved fluorescence assays, in which the immobilized antigen captures autoantibodies from the sample and detection is achieved using labeled antigen [12]–[14]. Even though these assays do not require radiolabeled compounds, commercially available ELISAs are relatively time-consuming and expensive and still need specialized equipment.…”

Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application

“…The protocol employed was based on those previously described [50, 59–61], with minor modifications. Briefly, polystyrene microplates (Maxisorp, NUNC, Roskilde, Denmark) were coated ON at 4 °C with 0.50 μg purified avidin per well, in coating buffer and washed five times with PBS.…”