A highly sensitive microsphere-based assay for early detection of Type I diabetes
Abstract
Type 1 Diabetes Mellitus (T1DM) is a T-cell mediated autoimmune disease in which deterioration of insulin producing pancreatic β-cells leads to a state of insulin deficiency. It has been shown that the clinical symptoms of T1DM are preceded by presence of islet cell autoantibodies (ICA) in serum. Radioimmunoassay (RIA) based detection of ICA are the current gold standard for diagnosis of T1DM. While the onset of hyperglycemia is an indicator of onset of T1DM, detection of ICA within the serum is important to differentiate T1DM from ketogenic Type 2 Diabetes (T2D) and Maturity Onset Diabetes of the Young (MODY). Due to their limited range of sensitivity, however, RIA cannot detect ICA at low concentrations in serum which could lead to delay in proper diagnosis and treatment. In addition, the use of radioactive species presents major disadvantages including exposure, waste removal, need for specialized licensed facilities to conduct the tests and the time required for the test (> 24 hours). To overcome these limitations, we have developed a rapid, highly sensitive, fluorescent and microsphere-based assay technique using Rolling Circle Amplification (RCA), to profile T1DM marker antibodies in serum. This assay utilizes the ability of RCA to detect very small amounts of DNA coupled with microsphere-immobilization resulting in an assay which is at least 50 times more sensitive than RIA. Further, this assay method requires very low volume of sample (5 μL), and can be easily adapted to detect other autoantibodies at similar sensitivities while reducing the assay time to ~6 hours. This powerful technique could enable detection of T1DM markers much earlier than current methods and enable earlier intervention to deter the progression of disease. In addition, the modularity of this assay would have implications for enhancing the sensitivities of any standard ELISA technique.
