Recombinant IA-2 expressed in E. coli can be used for the routine detection of autoantibodies in type-I diabetes

Morgenthaler, N. G. and Löbner, K. and Morgenthaler, U. Y. and Christie, M. R. and Seissler, J. and Scherbaum, W. A. (1998) Recombinant IA-2 expressed in E. coli can be used for the routine detection of autoantibodies in type-I diabetes. Hormone and Metabolic Research, 30 (9). pp. 559-564. ISSN 0018-5043

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Item Type:Article
Item Status:Live Archive

Abstract

We have investigated the possibility of measuring autoantibodies to IA- 2 (IA-2A) using recombinant protein expressed in E. coil in a new radioassay. The intracellular part of IA-2 (IA-2ic) was expressed in E. coil as a biotinylated fusion protein and affinity-purified on a streptavidin column. The average yield of IA2ic was about 1 mg purified protein from one litre of culture medium with E. coil. 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 contrast 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.

Keywords:autoantibody, biotin, hybrid protein, iodine 125, methionine, protein tyrosine phosphatase, recombinant protein, streptavidin, sulfur 35, adolescent, adult, aged, antibody detection, article, autoimmune disease, child, controlled study, escherichia coli, graves disease, human, human tissue, immunoprecipitation, insulin dependent diabetes mellitus, isotope labeling, major clinical study, priority journal, radioassay, serum, Adolescent, Antibody Specificity, Autoantibodies, Autoantigens, Child, Preschool, Diabetes Mellitus, Type 1, Epitopes, Gene Expression Regulation, Bacterial, Humans, Infant, Iodine Radioisotopes, Membrane Glycoproteins, Membrane Proteins, Middle Aged, Precipitin Tests, Protein-Tyrosine-Phosphatase, Radioligand Assay, Recombinant Proteins, Escherichia coli
Subjects:A Medicine and Dentistry > A100 Pre-clinical Medicine
Divisions:College of Science > School of Life Sciences
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ID Code:18142
Deposited On:10 Aug 2015 10:51

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