Diabetes Antibody Standardization Program: First Proficiency Evaluation of Assays for Autoantibodies to Zinc Transporter 8

Abstract

BACKGROUND Zinc transporter 8 (ZnT8) is a recently identified major autoantigen in type 1 diabetes, and autoantibodies to ZnT8 (ZnT8A) are new markers for disease prediction and diagnosis. Here we report the results of the first international proficiency evaluation of ZnT8A assays by the Diabetes Antibody Standardization Program (DASP). METHODS After a pilot workshop in 2007, an expanded ZnT8A workshop was held in 2009, with 26 participating laboratories from 13 countries submitting results of 63 different assays. ZnT8A levels were measured in coded sera from 50 patients with newly diagnosed type 1 diabetes and 100 blood donor controls. Results were analyzed comparing area under the ROC curve (ROC-AUC), sensitivity adjusted to 95% specificity (AS95), concordance of sample ZnT8A positive or negative designation, and autoantibody levels. RESULTS ZnT8A radio binding assays (RBAs) based on combined immunoprecipitation of the 2 most frequent ZnT8 COOH-terminal domain polymorphic variants showed a median ROC-AUC of 0.848 [interquartile range (IQR) 0.796–0.878] and a median AS95 of 70% (IQR 60%–72%). These RBAs were more sensitive than assays using as antigen either 1 ZnT8 variant only or chimeric constructs joining NH2- and COOH-terminal domains, assays based on immunoprecipitation and bioluminescent detection, or assays based on immunofluorescent staining of cells transfected with full-length antigen. CONCLUSIONS The DASP workshop identified immunoprecipitation-based ZnT8A assays and antigen constructs that achieved both a high degree of sensitivity and specificity and were suitable for more widespread clinical application.

Islet autoantibody phenotypes and incidence in children at increased risk for type 1 diabetes

“…Measurement of islet autoantibodies in these studies was performed using radiobinding assays (RBAs), as described elsewhere [9][10][11]. Islet autoantibody positivity was defined as the development of persistent autoantibodies against one or more of the antigens insulin, GAD65, IA-2 or ZnT8, with sample values above the 99th percentile of published population control children classified as positive.…”

“…Children were classified as multiple islet autoantibody-positive if they tested positive for more than one of the islet autoantibodies on at least one occasion. Islet autoantibody assays were evaluated according to the Diabetes Autoantibody Standardization Program [9][10][11] and type 1 diabetes was diagnosed according to ADA Expert Committee criteria [12].…”

Characteristics of rapid vs slow progression to type 1 diabetes in multiple islet autoantibody-positive children

“…The assays had sensitivities and specificities of 86% and 93% (GADA) [12], 72% and 100% (IA-2A) [12], 70% and 99% (IAA) [13], 68% and 100% (ZnT8RA) [14] and 51% and 100% (ZnT8WA) [14] in proficiency workshops. The interassay CVs for samples with a low autoantibody titre were 18% (GADA), 16% (IA-2A), 11% (IAA), 17% (ZnT8RA) and 16% (ZnT8WA), respectively.…”

“…Autoantibody assays were evaluated by the Diabetes Antibody Standardization Program, and the performances are shown as those of laboratory 121 in published reports [12][13][14]. The assays had sensitivities and specificities of 86% and 93% (GADA) [12], 72% and 100% (IA-2A) [12], 70% and 99% (IAA) [13], 68% and 100% (ZnT8RA) [14] and 51% and 100% (ZnT8WA) [14] in proficiency workshops.…”

Thyroid autoimmunity in relation to islet autoantibodies and HLA-DQ genotype in newly diagnosed type 1 diabetes in children and adolescents

“…Inter-assay CV for ZnT8RA was 7%, ZnT8WA 8% and ZnT8QA 10%. Our laboratory has been validated in Diabetes Autoantibody Standardization Program (DASP) 2011 with 50% study sensitivity and 100% study specificity for ZnT8RA, 46% study sensitivity and 100% study specificity for ZnT8WA and 38% study sensitivity and 100% study specificity for ZnT8QA [16]. Cut-off values were ZnT8RA ≥75, ZnT8WA ≥75 and ZnT8QA ≥100 U/ml.…”