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 hav e assessed autoantibody RIAs using a series of ICA512/IA-2 constructs to produce in vitro synthesized S-35-methionine-labeled proteins. Leve ls of ICA512/IA-2 (256-979, truncated aminoterminus) autoantibodies we re strongly correlated with those of the full-length ICA512/IA-2 (1-97 9) autoantibodies (r = 0.96, P < 0.0001) and ICA512/IA-2 (687-979) aut oantibodies (r = 0.98, P < 0.0001). RIAs using these 3 constructs had increased sensitivity relative to our initially reported ICA512 autoan tibody RIA (amino acids 389-948, truncated carboxy- and aminoterminus) . Only 2 of 38 sera examined in this study reacted with an aminotermin us ICA512/IA-2 (1-577) construct. The mean SD score (so score = (index of unknown sample mean index of controls) using of controls) using th e ICA512/IA-2 (256-979) construct was significantly higher than the sn score obtained with other ICA512/IA-2 constructs (P < 0.001). Amongst patients with new-onset diabetes and prediabetic relatives, using RIA s for autoantibodies reacting with ICA512/IA-2 (256-979), insulin, and glutamic acid decarboxylase 65, 98% expressed one or more of these au toantibodies and 78% expressed two or more, whereas no control (n = 20 8) expressed more than a single autoantibody. A combined ICA512/IA-2 ( 256-979), glutamic acid decarboxylase 65 autoantibody RIA with differe ntial autoantigen labeling (S-35-methionine, H-3-leucine) has been dev eloped that uses 96-well plate membrane filtration and Top Counter bet a counting. Concordance between results of dual and single RIAs was gr eater than 90%. This simple combined autoantibody assay should facilit ate large-scale autoantibody screening.