Islet tyrosine phosphatase 2 (IA-2) is one of the major autoantigens in type 1 diabetes. The aim of this work was to evaluate which IA-2 construct(s) among those usually employed has the highest sensitivity and specificity for detecting IA-2 autoantibodies in autoimmune diabetes and whether the combination of different IA-2 constructs into a single assay allows the detection of immunoreactivities otherwise not detectable by a single construct. For this purpose, we tested the single immunoreactivities of IA-2 FL(aa 1-979), IA-2(BDC)(aa 256-556:630-979), IA-2 IC(aa 605-979), IA-2(aa 256-760), IA-2(aa 761-928), and of 7 combinations of these fragments in the sera of 203 newly diagnosed type 1 diabetic patient (DM: 109 males,94 females, mean age 12.9 +/- 7.5 years) and 43 prediabetic subject (PDM: 20 males, 23 females, mean age 10.3 +/- 6.0 years) sera. IA-2 IC was the single construct that showed the highest sensitivity and specificity both in DM and PDM subjects; however, all of the other IA-2 constructs investigated detected additional immunoreactivities with respect to it. The combined use into the same assay of IA-2 IC, IA-2 FL, and IA-2 (256-760) constructs allowed detection of IA-2 Abs in additional 13.3% DM and 30.4% PDM subjects compared to the single IA-2 IC construct, suggesting this methodology as a new, highly sensitive approach to the study of IA-2 autoimmunity in type 1 diabetes.