Improved Specificity of Glutamate Decarboxylase 65 Autoantibody Measurement Using Luciferase-Based Immunoprecipitation System Assays

Abstract

Autoantibodies 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 Nanoluc luciferase (Nluc)-conjugated GAD65 constructs, Nluc-GAD65(96-585) and full length Nluc-GAD65(1-585), were evaluated in 434 well-characterized serum samples from patients with re­cent-onset type 1 diabetes and first-degree relatives. Non­radioactive, 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 GAD65 by 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 nonspecific 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.