GAD65 autoantibodies detected by electrochemiluminescence assay identify high risk for type 1 diabetes

Abstract

The identification of diabetes-relevant islet autoantibodies is essential for predicting and preventing type 1 diabetes (T1D). The aim of the current study was to evaluate a newly developed electrochemiluminescence (ECL)-GAD antibody (GADA) assay and compare its sensitivity and disease relevance with standard radioassay. The assay was validated with serum samples from 227 newly diagnosed diabetic children; 68 prediabetic children who were prospectively followed to T1D; 130 nondiabetic children with confirmed islet autoantibodies to insulin, GAD65, IA-2, and/or ZnT8 longitudinally followed for 12 ± 3.7 years; and 181 age-matched, healthy, antibody-negative children. The ECL-GADA assay had a sensitivity similar to that of the standard GADA radioassay in children newly diagnosed with T1D, prediabetic children, and high-risk children with multiple positive islet autoantibodies. On the other hand, only 9 of 39 nondiabetic children with only a single islet autoantibody (GADA only) by radioassay were positive for ECL-GADA. GADA not detectable by ECL assay is shown to be of low affinity and likely not predictive of future diabetes. In conclusion, the new ECL assay identifies disease-relevant GADA by radioassay. It may help to improve the prediction and correct diagnosis of T1D among subjects positive only for GADA and no other islet autoantibodies.

FIG. 1.

GADA levels from ECL-GADA assay compared with the standard NIDDK harmonized GADA radioassay. The dashed lines represent the assay cutoffs. The two assays were correlated (P < 0.0001) for all three groups, with both assays set at 99% specificity. A: Newly diagnosed children with T1D (n = 177) at the Barbara Davis Center. B: Prediabetic DAISY participants (n = 68). C: Newly diagnosed DASP workshop participants (n = 50).

FIG. 2.

A: GADA levels from ECL-GADA assay compared with the standard NIDDK harmonized GADA radioassay in all 39 DAISY participants who were persistently positive for single islet autoantibody (GADA only) on multiple clinic visits with years of follow-up. B: ECL-GADA results were consistently negative in all but 9 participants. The horizontal line represents the 3-SD score, which is close to the cutoffs for both assays.

FIG. 3.

A: GADA levels from ECL-GADA assay compared with the standard NIDDK harmonized GADA radioassay in all 43 DAISY participants who were persistently positive for multiple islet autoantibodies. B: GADA levels on ECL-GADA assay and GADA radioassay correlated closely. The horizontal line represents the 3-SD score, which is close to the cutoffs for both assays.

FIG. 4.

Sera with positive GADA by radioassay from 14 subjects (7 ECL-GADA positive and 7 ECL-GADA negative) were incubated with different concentrations of unlabeled GAD65 protein and analyzed with standard NIDDK harmonized GADA radioassay. GADA negative by ECL-GADA assay (dotted line), compared with GADA positive by ECL-GADA assay (solid line), required higher concentrations of GAD65 protein for 50% maximal inhibition, which is consistent with low affinity. Results are expressed as percentage of signal not absorbed.