AbstractAutoantibodies to glutamate decarboxylase (GADA) are widely used in the prediction and classification of type 1 diabetes. GADA radiobinding assays (RBAs) using N-terminally truncated antigens offer improved specificity but radioisotopes limit the high-throughput potential for population screening. Luciferase-based Immunoprecipitation System (LIPS) assays are sensitive and specific alternatives to RBAs with the potential to improve risk stratification.The performance of assays using the Luciferase (Nluc-) conjugated GAD65constructs, Nluc-GAD65(96-585) and full length Nluc-GAD65(1-585) were evaluated in 434 well-characterised sera from recent-onset type 1 diabetes patients and first-degree relatives.Non-radioactive, high-throughput LIPS assays are quicker and require less serum than RBAs. of 171 relatives previously tested single autoantibody positive for autoantibodies to full-length GAD65by RBA but had not progressed to diabetes, fewer retested positive by LIPS using either truncated (n=72) or full-length (n=111) antigen. The Nluc-GAD65(96-585) truncation demonstrated the highest specificity in LIPS assays overall but in contrast to RBA, N-terminus truncations did not result in a significant increase in disease-specificity compared with the full-length antigen. This suggests that binding of non-specific antibodies is affected by the conformational changes resulting from addition of the Nluc antigen. Nluc-GAD65(96-585) LIPS assays offer low blood volume, high specificity GADA tests for screening and diagnostics.

The diagnosis of diabetes type in adulthood can be difficult due to overlapping phenotypes and lack of clear classification guidelines. Type 1 and type 2 diabetes have very different treatment and care requirements, and incorrect classification can lead to life-threatening consequences. Islet autoantibodies are biomarkers of the autoimmune pathology of type 1 diabetes and can be used in prediction of risk and classification of diabetes type, however current tests have imperfect specificity and sensitivity, and there are a number of remaining questions for optimal use.
The overall aim of this thesis was to refine the use of islet autoantibody testing in the diagnosis of adult-onset diabetes by:
1. Exploring optimal test thresholds, and whether these are influenced by age.
2. Determining whether islet antibody level has potential utility in patients with clinically diagnosed type 1 diabetes.
3. Examining whether recently developed assays to specific GAD epitopes, isotype and affinity can improve test utility.
In Chapter 1 we provide an introduction to diabetes and the different types, and present some of the difficulties surrounding diabetes classification. Next, we provide a detailed background into islet autoantibodies and review the current evidence for their use in clinical practice and challenges in interpreting their results.
In Chapter 2, we looked at whether the use of age-related positivity thresholds are necessary to improve zinc transporter 8 autoantibody (ZnT8A) assay performance using the commercially available RSR ELISA assay. Our first key finding was that the prevalence of detectable ZnT8A differed between those tested under and over 30 years of age in the general population. ZnT8A age-related positivity thresholds improved the specificity of the assay whilst maintaining sensitivity, and that using one positivity threshold can result in misclassification of diabetes type.
In Chapter 3, we looked at the utility of islet autoantibody level at diagnosis of type 1 diabetes. Our main finding was a bimodal distribution of levels of glutamate decarboxylase (GADA) and islet antigen-2 autoantibodies (IA-2A) at diagnosis of type 1 diabetes, but not for ZnT8A. Those with high level GADA were older at diagnosis, more likely to be female and to be diagnosed with another autoimmune disease. In contrast, those with high level IA-2A were more likely to be younger at diagnosis and have ZnT8A as additional islet autoimmunity. This was replicated in a second cohort using an alternative method of islet autoantibody assessment. These findings increased our understanding of how islet autoantibody levels at diagnosis are associated with differences in the underlying pathology between age groups at diabetes diagnosis.
In Chapter 4 we looked at GADA epitope specificity, affinity and IgG subclass response in those positive for GADA, clinically diagnosed with type 2 diabetes in adult-hood. In full-length (f-)GADA positive adult-onset diabetes, our novel finding was that testing for truncated (t-)GAD(96-585) autoantibodies stratified risk of progression to early insulin therapy (within 5 years) and identified those with a more type 1 diabetes-like phenotype; lower C-peptide, higher type 1 diabetes genetic susceptibility and positivity for IA-2A. In contrast, testing for f-GADA affinity and IgG subclass response did not stratify risk of progression to early insulin requirement. These findings provide evidence to support the testing for t-GADA in adult-onset patients.
In Chapter 5, we summarize the key findings, the limitations of this work and its implications. Then we present ideas for future research and how to take these findings further.
In summary, here we have provided evidence for the improvement of islet autoantibody testing by taking three different approaches. Firstly, we have shown how using age-related cut-offs improves specificity of a commercially available assay and how these thresholds reduce the risk of misclassification. Secondly, that some differences between child- and adult-onset diabetes are associated with islet autoantibody level and thirdly, that testing for GADA epitope specificity in adult-onset diabetes, can help predict who will require early insulin therapy. Implementation of these findings in clinical testing will improve outcomes for individuals with diabetes.

CONTEXT: the importance of the autoantibody level at diagnosis of type 1 diabetes (T1D) is not clear. OBJECTIVE: We aimed to assess the association of glutamate decarboxylase (GADA), islet antigen-2 (IA-2A), and zinc transporter 8 (ZnT8A) autoantibody levels with clinical and genetic characteristics at diagnosis of T1D. METHODS: We conducted a prospective, cross-sectional study. GADA, IA-2A, and ZnT8A were measured in 1644 individuals with T1D at diagnosis using radiobinding assays. Associations between autoantibody levels and the clinical and genetic characteristics for individuals were assessed in those positive for these autoantibodies. We performed replication in an independent cohort of 449 people with T1D. RESULTS: GADA and IA-2A levels exhibited a bimodal distribution at diagnosis. High GADA level was associated with older age at diagnosis (median 27 years vs 19 years, P = 9 × 10-17), female sex (52% vs 37%, P = 1 × 10-8), other autoimmune diseases (13% vs 6%, P = 3 × 10-6), and HLA-DR3-DQ2 (58% vs 51%, P =. 006). High IA-2A level was associated with younger age of diagnosis (median 17 years vs 23 years, P = 3 × 10-7), HLA-DR4-DQ8 (66% vs 50%, P = 1 × 10-6), and ZnT8A positivity (77% vs 52%, P = 1 × 10-15). We replicated our findings in an independent cohort of 449 people with T1D where autoantibodies were measured using enzyme-linked immunosorbent assays. CONCLUSION: Islet autoantibody levels provide additional information over positivity in T1D at diagnosis. Bimodality of GADA and IA-2A autoantibody levels highlights the novel aspect of heterogeneity of T1D. This may have implications for T1D prediction, treatment, and pathogenesis.

IntroductionZinc transporter 8 autoantibodies (ZnT8A) are biomarkers of beta cell autoimmunity in type 1 diabetes that have become more widely available to clinicians in recent years. Robust control population-defined thresholds are essential to ensure high clinical specificity in islet autoantibody testing. We aimed to determine the optimal cut-offs for ZnT8A testing.Research design and methods97.5th and 99th centile cut-offs were determined using residual clinical sera from 1559 controls aged between 0 and 83 years with no history of diabetes and a hemoglobin A1c level of less than 6.0% (<42 mmol/mol). ZnT8A were measured by ELISA (RSR, Cardiff, UK) on a Dynex DS2 ELISA robot (Dynex, Preston, UK). We assessed the impact of age-related cut-offs in comparison with the manufacturer’s recommended threshold in a mixed cohort of young-onset (<age 30) diabetes (UNITED study (Using pharmacogeNetics to Improve Treatment in Early-onset Diabetes), n=145).ResultsUsing the manufacturer’s limit of detection, 6 WHO U/mL, 16.2% of people in the control cohort had detectable levels of ZnT8A and those who had detectable ZnT8A were much more likely to be younger (p<0.0001). The 97.5th and 99th centile thresholds were substantially higher in younger participants: 18 and 127 WHO U/mL (tested under 30 years) in comparison with 9 and 21 WHO U/mL (tested 30 years and over). In the UNITED cohort some of those found to be ZnT8A-positive by the manufacturer’s threshold but negative using the appropriate 99% centile cut-off (127 WHO U/mL) displayed characteristics suggestive of type 2 diabetes.ConclusionsAge-related thresholds are needed for ZnT8A testing. In those aged <30 years, use of manufacturers’ recommended cut-offs may result in low test specificity and potentially high rates of false positive test results in patients who do not have autoimmune diabetes.