Capillary Electrophoresis Immunoassay for Detection of Anti-insulin Antibodies

Type 1 diabetes mellitus is an autoimmune disorder primarily affecting children and adolescents. Diagnostic techniques for autoimmune diseases typically examine the amounts of autoantibodies (AAbs) and are performed using enzyme-linked immunosorbent assays and radioimmunoassays. Insulin autoantibody (IAA) is one of the earliest biomarkers in juveniles prone to type 1 diabetes; therefore, the development of highly sensitive assays could enhance early diagnostic capabilities. To this end, we developed a capillary electrophoresis laser-induced fluorescence noncompetitive immunoassay for the detection of monoclonal anti-insulin Abs as a first step toward the development of an IAA assay. In this study, a fluorescently labeled insulin was used as an affinity probe to target anti-insulin Abs. In the presence of an Ab, the separated mixture showed two distinct peaks correlating to bound and free labeled insulin. Sample incubation and separation parameters were optimized, and produced a detection limit (LOD) of 1.5 ng/mL (∼10 pM) of Ab. To further demonstrate the utility of the assay, calibration in a serum matrix was also performed and showed a linear response. The assay was also tested using a polyclonal Ab, which resulted in a dose-dependent decrease in the labeled insulin peak, producing an LOD of 170 ng/mL (∼1 nM). This robust system provides a foundation for further development of an IAA assay.