Development of predictive assay for progression of slow-onset type 1 diabetes and its application to disease prevention

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Japanese type 1 diabetes (T1D) is a heterogeneous disease characterized by the different clinical features in terms of its mode of onset or age at disease onset. It is estimated that the prevalence of adult-onset T1D is similar to that of childhood-onset T1D, and that around two-thirds of adult-onset T1D are categorized in "slow-onset T1D". Although anti-glutamic acid decarboxylase 65 (GAD65) antibodies are good predictive marker for future insulin deficiency in slow-onset T1D, positive predictive value at 5 years is about 65% and one-thirds of GAD65 antibody-positive patients do not need insulin therapy for long period. Therefore, in order to develop the highly predictive marker for future insulin deficiency in slow-onset T1D we performed a fine epitope mapping of anti-AD65 antibodies using random phage display technology.1. Preparation of human islet GAD random phage display libraryIn the first year we prepared a human islet GAD random phage display library, and screened this library with pooled sera from patients with slow-onset T1D and healthy control subjects. After amplifying the individual clone that reacted with sera from patients or controls, the PCR-direct sequence analysis was performed to determine the specific DNA sequence for GAD65 molecule.2. Development of anti-GAD epitope-specific antibody measurementWith the results obtained in the first year, we tried to identify the specific epitopes associated with the progression to insulin deficiency in slow-onset T1D patients by sandwich ELISA method. There were many difficulties to identify the diabetes-specific epitopes for GAD antibodies using random phage display library system with ELISA because of the high Noise/Signal ratio.We are trying to do the fine epitope mapping using other detection systems.