Recombinant IA-2 expressed in E. coli can be used for the routine detection of autoantibodies in Type-I diabetes.
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.
Morgenthaler
N G
NG
Department of Medicine III, University of Leipzig, Germany. morgenthaler@brahms.de
Löbner
K
K
Morgenthaler
U Y
UY
Christie
M R
MR
Seissler
J
J
Scherbaum
W A
WA
D000293:Adolescent; D000328:Adult; D000368:Aged; D000918:Antibody Specificity; D001323:Autoantibodies / Q000097:blood / Q000302:isolation & purification*; D001324:Autoantigens / Q000235:genetics / Q000276:immunology*; D002648:Child; D002675:Child, Preschool; D003922:Diabetes Mellitus, Type 1 / Q000150:complications / Q000201:enzymology / Q000276:immunology*; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000276:immunology; D006111:Graves Disease / Q000150:complications / Q000276:immunology; D006801:Humans; D007223:Infant; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology*; D008565:Membrane Proteins; D008875:Middle Aged; D011233:Precipitin Tests; 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; D011994:Recombinant Proteins / Q000276:immunology*
D001323:Autoantibodies; D001324:Autoantigens; D000939:Epitopes; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C102889:PTPRN2 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8