Detection of autoantibodies against reactive oxygen species modified glutamic acid decarboxylase-65 in type 1 diabetes associated complications

Abstract

Background

Autoantibodies against glutamate decarboxylase-65 (GAD ₆₅Abs) are thought to be a major immunological tool involved in pathogenic autoimmunity development in various diseases. GAD ₆₅Abs are a sensitive and specific marker for type 1 diabetes (T1D). These autoantibodies can also be found in 6-10% of patients classified with type 2 diabetes (T2D), as well as in 1-2% of the healthy population. The latter individuals are at low risk of developing T1D because the prevalence rate of GAD ₆₅Abs is only about 0.3%. It has, therefore, been suggested that the antibody binding to GAD ₆₅ in these three different GAD ₆₅Ab-positive phenotypes differ with respect to epitope specificity. The specificity of reactive oxygen species modified GAD ₆₅ (ROS-GAD ₆₅) is already well established in the T1D. However, its association in secondary complications of T1D has not yet been ascertained. Hence this study focuses on identification of autoantibodies against ROS-GAD ₆₅ (ROS-GAD ₆₅Abs) and quantitative assays in T1D associated complications.

Results

From the cohort of samples, serum autoantibodies from T1D retinopathic and nephropathic patients showed high recognition of ROS-GAD ₆₅ as compared to native GAD ₆₅ (N-GAD ₆₅). Uncomplicated T1D subjects also exhibited reactivity towards ROS-GAD ₆₅. However, this was found to be less as compared to the binding recorded from complicated subjects. These results were further proven by competitive ELISA estimations. The apparent association constants (AAC) indicate greater affinity of IgG from retinopathic T1D patients (1.90 × 10 ^-6 M) followed by nephropathic (1.81 × 10 ^-6 M) and uncomplicated (3.11 × 10 ^-7 M) T1D patients for ROS-GAD ₆₅ compared to N-GAD ₆₅.

Conclusion

Increased oxidative stress and blood glucose levels with extended duration of disease in complicated T1D could be responsible for the gradual formation and/or exposing cryptic epitopes on GAD ₆₅ that induce increased production of ROS-GAD ₆₅Abs. Hence regulation of ROS-GAD ₆₅Abs could offer novel tools for analysing and possibly treating T1D complications.