Improved Specificity of Glutamate Decarboxylase 65 Autoantibody Measurement Using Luciferase-Based Immunoprecipitation System Assays
Rebecca C. Wyatt, Sian Grace, Cristina Brigatti, Ilaria Marzinotto, Benjamin T. Gillard, Deborah K. Shoemark, Kyla Chandler, Peter Achenbach, Lorenzo Piemonti, Anna E. Long, Kathleen M. Gillespie, Vito Lampasona, Alistair J.K. Williams, BOX Study Group, Isabel Wilson, Rachel J. Aitken, Ilana Kelland, Clare Megson, Chitrabhanu Ballav, Atanu Dutta, Michelle Russell‐Taylor, Rachel Besser, James Bursell, Shanthi Chandran, Sejal Patel, Anne Smith, Manohara Kenchaiah, Gomathi Margabanthu, Foteini Kavvoura, Chandan Yaliwal
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.