GAD Autoantibody Affinity and Epitope Specificity Identify Distinct Immunization Profiles in Children at Risk for Type 1 Diabetes

Abstract

OBJECTIVE—Autoantibodies to insulin and GAD are features of preclinical type 1 diabetes in children. For insulin autoantibodies, the antibody affinity and epitope specificity predict which children progress to diabetes. We asked whether autoantibodies to GAD (GADAs) are heterogeneous in affinity and epitope recognition and whether diabetes-related GADA are restricted to high-affinity responses. RESEARCH DESIGN AND METHODS—GADA affinity was measured by competitive binding experiments with [125I]-labeled and -unlabeled recombinant human GAD65 in the first GADA-positive sample from 95 children with a type 1 diabetes family history who were prospectively followed from birth and in follow-up samples from 65 of these children. RESULTS—At first GADA appearance, affinity ranged from 107 to 1010 l/mol. Affinity was higher in multiple islet autoantibody-positive children (P < 0.0001) and in HLA DR3–positive children (P = 0.006). GADA affinities were >109 l/mol in 52 of 53 multiple autoantibody-positive children. In contrast, children who were single GADA positive often had lower affinity GADA and/or GADA with specificities that were restricted to minor NH2-terminal GAD65 epitopes. At follow-up, affinity increased from low to high in 3 of 65 children. All 24 children who developed diabetes had high-affinity GADAs before diabetes onset. CONCLUSIONS—Children develop discrete, heterogeneous antibody responses to GAD that could arise from distinct immunization events, only some of which are diabetes relevant. Subtyping the GADA responses using affinity measurement will improve type 1 diabetes risk assessment.

Slowly progressive insulin-dependent (type 1) diabetes positive for anti-GAD antibody ELISA test may be strongly associated with a future insulin-dependent state

“…In fact, it is believed that the bridging conformation favors the recognition of high-affinity antibodies, contributing to the good specificity [14]. Given that researches say that the existence of high-affinity GADA is more closely associated with the progression of type 1 DM [15,16], the possible high disease activity in GADA-ELISA-positive and GADA-RIA-positive SPIDDM is easily understandable.…”

GAD autoantibody affinity in schoolchildren from the general population

“…The reproducibility of GADA affinity measurements was determined from replicates of a GADA-positive serum that was included in each experiment. The CV of GADA affinity measurements was 3% [14]. The calculation of K d values was limited to samples with IC 50 values greater than the concentration of labelled GAD65 (0.05 nmol/l).…”

“…GADA affinity measurements GADA affinity was measured by competitive binding experiments as previously described [14]. Briefly, serum (2 μl) was incubated in duplicate for 48 h at 4ºC in TBST buffer (50 mol/l Tris, 150 mmol/l NaCl, 1% Tween 20, pH 7.4) in the presence of [ 125 I]-recombinant human GAD65 (0.05 nmol/l) and five increasing quantities of unlabelled human GAD65 (3.3×10 −11 , 3.3×10 −10 , 3.3× 10 −9 , 3.3×10 −8 and 3.3×10 −7 mol/l) (RSR Ltd., Cardiff, UK) or TBST buffer only in a final volume of 47 μl.…”

“…GADA often appear in older age groups and are preferentially associated with HLA-DRB1*03 haplotypes [11]. The GADA level alone does not predict progression to diabetes, and controversial reports exist with respect to the relevance of GADA epitope reactivity to diabetes progression [12][13][14][15][16][17].…”

“…We previously demonstrated that children with a familial predisposition who developed type 1 diabetes had highaffinity GADA before the onset of their disease, whereas those with low-affinity GADA showed no progression of islet autoimmunity [14]. Nevertheless, approximately 90% of children diagnosed with type 1 diabetes have no first-degree relatives with the disease [18][19][20].…”