Improving clinical utility of GAD65 autoantibodies by electrochemiluminescence assay and clinical phenotype when identifying autoimmune adult-onset diabetes

Abstract

Aims/hypothesis: It is important to differentiate the two major phenotypes of adult-onset diabetes, autoimmune type 1 diabetes and non-autoimmune type 2 diabetes, especially as type 1 diabetes presents in adulthood. Serum GAD65 autoantibodies (GADA) are the most sensitive biomarker for adult-onset autoimmune type 1 diabetes, but the clinical value of GADA by current standard radiobinding assays (RBA) remains questionable. The present study focused on the clinical utility of GADA differentiated by a new electrochemiluminescence (ECL) assay in patients with adult-onset diabetes.

Methods: Two cohorts were analysed including 771 diabetic participants, 30-70 years old, from the Action LADA study (n = 6156), and 2063 diabetic participants, 20-45 years old, from the Diabetes in Young Adults (DiYA) study. Clinical characteristics of participants, including requirement of early insulin treatment, BMI and development of multiple islet autoantibodies, were analysed according to the status of RBA-GADA and ECL-GADA, respectively, and compared between these two assays.

Results: GADA was the most prevalent and predominant autoantibody, >90% in both cohorts. GADA positivity by either RBA or ECL assay significantly discriminated clinical type 1 from type 2 diabetes. However, in both cohorts, participants with ECL-GADA positivity were more likely to require early insulin treatment, have multiple islet autoantibodies, and be less overweight (for all p < 0.0001). However, clinical phenotype, age at diagnosis and BMI independently improved positive predictive value (PPV) for the requirement of insulin treatment, even augmenting ECL-GADA. Participants with GADA detectable by RBA, but not confirmed by ECL, had a phenotype more similar to type 2 diabetes. These RBA-GADA positive individuals had lower affinity GADA compared with participants in which GADA was confirmed by ECL assay.

Conclusions/interpretation: Detection of GADA by ECL assay, given technical advantages over RBA-GADA, identified adult-onset diabetes patients at higher risk of requiring early insulin treatment, as did clinical phenotype, together allowing for more accurate clinical diagnosis and management.

Keywords: Adult-onset diabetes; Autoantibodies; Biomarker; ECL assay.

Fig. 1

Participants studied from the Action LADA and DiYA studies

Fig. 2

Overall IAb positivity including GADA, IA-2A and ZnT8A for the participants studied in the Action LADA study (a) and the DiYA study (b)

Fig. 3

Comparison of GADA levels between RBA and ECL assay in the (a) Action LADA study (n = 771) and (b) DiYA study (n = 2063). The solid dots represent individuals positive for IA-2A and/or ZnT8A and the open dots negative for IA-2A and ZnT8A. The dotted lines indicate assay cut-offs. RBA-GADA was tested at the Blizard Institute laboratory, London, for the Action LADA study, with a cut-off value of 68 (index), and at the Barbara Davis Center for Diabetes laboratory, USA, for the DiYA study, with a cut-off value of 20 (DK units). The ECL-GADA assay was performed at the Barbara Davis Center for Diabetes for both Action LADA and DiYA studies, with a cut-off value of 0.023 (index)

Fig. 4

Clinical features were compared in four groups of participants with adult-onset diabetes from the Action LADA study: RBA-GADA+ confirmed by ECL assay, RBA-GADA+ not confirmed by ECL assay, ECL-GADA+ only, and GADA– in both RBA and ECL assay. (a) BMI values; (b) per cent of participants requiring early insulin treatment; (c) per cent of participants positive for multiple IAbs. *p < 0.05, ***p < 0.001 by ANOVA test or logistic regression analyses

Fig. 5

Clinical features were compared in four groups of participants with adult-onset diabetes from the DiYA study: RBA-GADA+ confirmed by ECL assay, RBA-GADA+ not confirmed by ECL assay, ECL-GADA+ only, and GADA– by both RBA and ECL assay. (a) BMI values; (b) per cent of participants requiring early insulin treatment; (c) per cent of participants positive for multiple IAbs. *p < 0.05, ***p < 0.001 by ANOVA test or logistic regression analyses

Fig. 6

GADA affinity analysis. In total, 43 serum samples positive for RBA-GADA, including 17 samples from the Action LADA study (blue; nine confirmed by ECL assay and eight not confirmed by ECL assay) and 26 samples from the DiYA study (red; 18 confirmed by ECL assay and eight not confirmed by ECL assay) were analysed using competitive RBA and incubation with different concentrations of native GAD65 protein. GADA not confirmed by ECL assay (dotted line), compared with GADA confirmed by ECL assay (solid line), required higher concentrations of GAD65 protein for 50% maximal inhibition. Results were expressed as per cent of signal not absorbed