IA-2var AAb

The IA-2var full-length molecule was in vitro transcribed and translated in the presence of [³⁵S]-methionine (PerkinElmer) using the TNT coupled rabbit reticulocyte system (Promega, Madison, WI). AAb against IA-2var were detected by a modified quantitative radioimmunoprecipitation assay (11) using 50% protein A-Sepharose to separate free [³⁵S]-methionine from antibody-bound labeled products. Assays were run in triplicate, and the results were calculated as an index as previously described (19,20). The threshold for the IA-2var AAb assay was established as the 99th percentile of the AAb indexes using 178 healthy control sera (106 females and 72 males, mean age 34.6 ± 10.2 years, 76% Caucasian, cutoff index: 0.218). All AAb test results using the same samples were confirmed. The interassay variation for IA-2var AAb was 8.3% (n = 10), while the intra-assay variation was 2.4% (n = 15). The IA-2var AAb assay achieved ratings of 62% sensitivity and 99% specificity during testing for the 2016 Islet Autoantibody Standardization Program (IASP).