Evaluation of islet cell antigen (ICA) 512/IA-2 autoantibody radioassays using overlapping ICA512/IA-2 constructs.
Islet cell antigen (ICA) 512 also termed IA-2 is a novel autoantigen of type 1 diabetes, which has a tyrosine phosphatase-like domain. We have assessed autoantibody RIAs using a series of ICA512/IA-2 constructs to produce in vitro synthesized 35S-methionine-labeled proteins. Levels of ICA512/IA-2 (256-979, truncated aminoterminus) autoantibodies were strongly correlated with those of the full-length ICA512/IA-2 (1-979) autoantibodies (r = 0.96, P < 0.0001) and ICA512/IA-2 (687-979) autoantibodies (r = 0.98, P < 0.0001). RIAs using these 3 constructs had increased sensitivity relative to our initially reported ICA512 autoantibody RIA (amino acids 389-948, truncated carboxy- and aminoterminus). Only 2 of 38 sera examined in this study reacted with an aminoterminus ICA512/IA-2 (1-577) construct. The mean SD score (SD score = (index of unknown sample-mean index of controls)/SD of controls) using the ICA512/IA-2 (256-979) construct was significantly higher than the SD score obtained with other ICA512/IA-2 constructs (P < 0.001). Amongst patients with new-onset diabetes and prediabetic relatives, using RIAs for autoantibodies reacting with ICA512/IA-2 (256-979), insulin, and glutamic acid decarboxylase 65, 98% expressed one or more of these autoantibodies and 78% expressed two or more, whereas no control (n = 208) expressed more than a single autoantibody. A combined ICA512/IA-2 (256-979), glutamic acid decarboxylase 65 autoantibody RIA with differential autoantigen labeling (35S-methionine, 3H-leucine) has been developed that uses 96-well plate membrane filtration and Top Counter beta counting. Concordance between results of dual and single RIAs was greater than 90%. This simple combined autoantibody assay should facilitate large-scale autoantibody screening.
Kawasaki
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Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Denver 80262, USA.
Yu
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L
Gianani
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Verge
C F
CF
Babu
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Bonifacio
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E
Eisenbarth
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GS
D000293:Adolescent; D000328:Adult; D000368:Aged; D001323:Autoantibodies / Q000032:analysis*; D001324:Autoantigens; D002648:Child; D002675:Child, Preschool; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D005968:Glutamate Decarboxylase / Q000276:immunology; D006801:Humans; D007223:Infant; D007328:Insulin / Q000276:immunology; D008297:Male; D008565:Membrane Proteins / Q000276:immunology*; D008875:Middle Aged; D010446:Peptide Fragments / Q000235:genetics; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000276:immunology*; D011863:Radioimmunoassay; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8
D001323:Autoantibodies; D001324:Autoantigens; D018076:DNA, Complementary; D007328:Insulin; D008565:Membrane Proteins; D010446:Peptide Fragments; C516862:PTPRN protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D005968:Glutamate Decarboxylase