Recombinant IA-2 Expressed inE. colican be used for the Routine Detection of Autoantibodies in Type-I Diabetes

Abstract

We have investigated the possibility of measuring autoantibodies to IA-2 (IA-2A) using recombinant protein expressed in E. coli in a new radioassay. The intracellular part of IA-2 (IA-2ic) was expressed in E. coli as a biotinylated fusion protein and affinity-purified on a streptavidin column. The average yield of IA-2ic was about 1 mg purified protein from one litre of culture medium with E. coli. We could demonstrate the immunological activity of this material by blocking the autoantibody reactivity to in vitro synthesised IA-2ic. The IA-2ic fusion protein was then radiolabelled with 125I, purified by HPLC, and used in an immunoprecipitation assay for the detection of IA-2A. Sera from 46 of 68 (67%) patients with Type-I diabetes were positive by this radioassay, in contrst to only 2 of 50 (4%) patients with autoimmune thyroid disease and 1 of 114 (1 %) controls. There was a correlation between this radioassay and the previously established radioligand assay using synthesized 35S-methionine-labelled IA-2ic in vitro (r = 0.79, p < 0.001). We conclude that E. coli-derived IA-2 has the correct immunogenic conformation, and can be used for the detection of IA-2A with a similar sensitivity and specificity as the validated radioligand assay. This new assay can facilitate the measurement of IA-2A in routine laboratories where the radioligand assay is inconvenient or not available.

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

“…This chemical ligand binds selectively to vicinal dithiols, such as the ones present in the catalytic site of Trx (binding motif Cys-Gly-Pro-Cys). The levels of soluble protein expression obtained in E.coli GI724 were higher than those previously reported in the literature for prokaryotic systems [72, 73]. High purity levels were achieved, even higher than those reported in the results section (72–77 %), which does not include the contribution of TrxIA-2 ic dimer.…”

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 some studies, GAD65-IA-2ic fusion proteins have been used for the detection of GAD65 autoantibodies (GADAs) and IA-2As by RBA (8,9 ). 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 ).…”

Age-Specific Levels of Diabetes-Related GAD and IA-2 Antibodies in Healthy Children and Adults

“…Antibodies to the overexpressed intracellular domain of tyrosine phosphatase IA-2 (IA-2ic) in E. coli were measured by immunoprecipitation (BRAHMS Diagnostica GmbH, Berlin, Germany) as previously described 7 . Briefly, 20 μΐ serum were incubated overnight with 200 μΐ i2> I-labelled IA-2.…”