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Evaluation of Islet Cell Antigen (ICA) 512/IA-2 Autoantibody Radioassays Using Overlapping ICA512/IA-2 Constructs¹

Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center (E.K., L.Y., R.G., C.F.V., S.B., G.S.E.), Denver, Colorado 80262; and Department of Medicine I, Istituto Scientifico San Raffaele, University of Milan (E.B.), Milan, Italy 20132

Address all correspondence and requests for reprints to: George S. Eisenbarth, M.D., Ph.D., Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Box B-140, 4200 East 9th Avenue, Denver, Colorado 80262. E-mail: George.Eisenbarth{at}UCHSC.edu

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 ß counting. Concordance between results of dual and single RIAs was greater than 90%. This simple combined autoantibody assay should facilitate large-scale autoantibody screening.

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