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Improved specificity of glutamate decarboxylase 65 autoantibody measurement using luciferase-based immunoprecipitation system (LIPS) assays

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Version 2 2024-03-04, 18:41
Version 1 2024-01-16, 20:41
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posted on 2024-03-04, 18:41 authored by Rebecca C. Wyatt, Sian L. Grace, Cristina Brigatti, Ilaria Marzinotto, Ben T. Gillard, Deborah K. Shoemark, Kyla Chandler, Peter Achenbach, Lorenzo Piemonti, Anna E. Long, Kathleen M. Gillespie, Vito Lampasona, Alistair J.K. Williams, The BOX Study Group

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 Luciferase (Nluc-) conjugated GAD65 constructs, Nluc-GAD65(96-585) and full length Nluc-GAD65(1-585) were evaluated in 434 well-characterised sera from recent-onset type 1 diabetes patients and first-degree relatives. Non-radioactive, 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 non-specific 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.

Funding

This study was funded by a collaborative JDRF grant to KMG, AEL, AJKW, PA and VL (2-SRA-2020-964-S-B). RCW was funded by a University of Bristol PhD Scholarship. SLG is funded by a Diabetes UK grant (22/0006452). AEL is jointly funded as a Diabetes UK & JDRF RD Lawrence Fellow (18/0005778 and 3-APF-2018-591-A-N). The Bart’s Oxford study was funded by Diabetes UK (14/0004472). DKS was funded by BrisSynBio, the BBSRC and EPSRC Synthetic Biology Research Centre (BB/L01386X/1).

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