By 6 years of follow-up, approximately 40% of subjects in the former group, compared with 7% in the latter, had progressed to clinical T1DM. of insulin monoclonal antibody. The AUY922 (Luminespib, NVP-AUY922) assay cutoff index of 0.006 was set at the 99th percentile of 100 healthy controls, and the interassay coefficient of variation AUY922 (Luminespib, NVP-AUY922) was 4.8% ( em n /em =20). In the recent IASP Workshop at the 2013 AUY922 (Luminespib, NVP-AUY922) Immunology of Diabetes Society meeting (Lorne, Australia), among patients with newly diagnosed T1DM, the sensitivity for ECL-IAA was 68%, and the specificity was 99%. ECL-GADA assay The ECL-GADA assay method has also been previously published.13 In brief, 20?L of fivefold diluted serum was mixed with 20?L of antigen buffer containing both sulfo-tag and biotin-labeled GAD65 and incubated at 4C for overnight. The antigenCantibody complexes containing biotin were captured by a streptavidin-coated plate and counted on an Imager 2400. The results were expressed as an index against our internal standard positive control of GAD65 monoclonal antibody. The assay cutoff index of 0.023 was set at the 99.5th percentile of 181 healthy controls, and the interassay coefficient of variation was 8.8% ( em n /em =10). In the recent IASP Workshop at the 2013 Immunology of Diabetes Society meeting, among patients with newly diagnosed T1DM, the sensitivity for ECL-GADA was 76%, and the specificity was 99%. Other laboratory measurements The RADs for IAA, GADA, IA-2, and ZnT8A used in the present AUY922 (Luminespib, NVP-AUY922) study were all performed in our laboratory and have been previously published.14 HLA-DQ and DR polymorphisms were determined by allele-specific oligonucleotide genotyping.15 The haplotypes of interest were DQA1*0501-DQB1*0201 (DQ2), DQA1*0301-DQB1*0302 (DQ8), and DQA1*0102-DQB1*0602 (DQ6). Statistical analysis Statistical analyses were performed using correlation analysis, rank sum, or Fisher’s exact test in PRISM version 4.0 software (GraphPad Software Inc., San Diego, CA). Survival analyses used the log rank test in SAS version 9.3 software (SAS Institute, Cary, NC). Statistical significance was determined by an level of 0.05. Results ECL-IAA and -GADA assays improve assessment of T1DM risk Among 384 pre-T1DM subjects who were followed to T1DM, the sensitivity of the ECL-IAA assay (54.7%, 210/384) was similar to that for the RAD-IAA assay (48.4%, 186/384). The sensitivity of the ECL-GADA (79.2%, 304/384) and RAD-GADA (76.8%, 295/384) assays was also similar. Figure 1 compares positivity and levels of ECL-IAA and RAD-IAA (Fig. 1A) as well as ECL-GADA and RAD-GADA (Fig. 1B) among 384 prediabetes samples with specificity set at the 99th percentile for each assay. The levels were strongly correlated between ECL-IAA and RAD-IAA ( em R /em 2=0.55; em P /em 0.0001) and between ECL-GADA and RAD-GADA ( em R /em 2=0.70; em P /em 0.0001). Only nine RAD-IAA-positive and 16 RAD-GADA-positive samples at low levels were found to be negative in ECL assays. Open in a separate window FIG. 1. Comparison of islet autoantibody levels between electrochemiluminescence (ECL) and radioassay (RAD) among 384 TrialNet prediabetes subjects who were prospectively followed up to clinical type 1 diabetes mellitus. The dotted lines represent the assay cutoffs. All assays were set at 99% specificity. (A) ECL-insulin autoantibody (IAA) versus RAD-IAA with correlation em R /em 2=0.5480 and em P /em 0.0001. (B) ECL-glutamic acid decarboxylase 65 autoantibody (GADA) versus RAD-GADA with correlation em R /em 2=0.7047 and em P /em 0.0001. (Color graphics available online at www.liebertonline.com/dia) Of 3,100 subjects who had not developed T1DM, 2,298 were positive for at least one iAb, and 802 were negative for all iAbs. Among iAb-positive subjects, 75.2% (1,727/2,298) were positive for a single iAb; most were positive for IAA ( em n /em =421) or GADA ( em n /em =1,169) only. It is remarkable that both ECL-IAA and ECL-GADA assays discriminated high-risk subjects with multiple iAbs or those followed to T1DM from low-risk subjects with a single iAb. The results of the ECL-IAA assay in all subjects participating in the present study are summarized in Figure 2. Only 1 1.0% (8/802) of subjects negative for all iAb by radioassays were ECL-IAA positive. The iAb-positive subjects who had not progressed to T1D were subdivided into RAD-IAA negative ( em n /em =1,581) or RAD-IAA positive ( em n /em =717). Among the former, 4.1% (54/1,306) of subjects with a single iAb were ECL-IAA positive versus 19.0% (52/275) of subjects with multiple iAbs ( em P /em 0.0001); among the latter, 21.1% (89/421) of subjects with a single iAb (RAD-IAA) were ECL-IAA positive versus 88.5% Rabbit polyclonal to ANAPC10 (262/296) of subjects with multiple iAbs ( em P /em 0.0001). ECL-IAA positivity was found in 54.7% (210/384) of subjects who later developed diabetes (pre-T1DM), compared with 48.4% (186/384) for RAD-IAA positivity ( em P /em =0.097). Open in a separate window FIG. 2. Electrochemiluminescence-insulin autoantibody (IAA) results for all 3,484 TrialNet subjects: all four islet autoantibodies negative (AbC) ( em n /em =802); islet autoantibodies positive with radioassay (RAD)-IAA negative ( em n /em =1,581), subdivided into single islet autoantibody (1Ab) positive ( em n /em =1,306) and multiple islet autoantibodies (2Ab) positive ( em n /em =275); islet autoantibodies positive with RAD-IAA-positive ( em n /em =717),.