Objective
We propose to implement on the high throughput LIAISON CLIA platform (DiaSorin) autoantibody assays for GADA, IA-2A, ZnT8A and IAA. In particular we will:
• Design novel recombinant T1D antigens with tags compatible with capture on the paramagnetic particles used in the LIAISON platform
• Validate these antigens before transfer to CLIA using liquid phase binding LIPS assays
• Develop and optimize CLIA protocol to enhance assay sensitivity and specificity.
• Validate the newly developed CLIA assays by testing cohorts of T1D and control sera
Background Rationale
The gold standard immunoassay format for the measurement of T1D autoantibodies is the Radio Binding Assay (RBA) a liquid phase binding immunoassays that uses recombinant radioactive antigens as tracers. RBA has been in use for decades and its performance is still among the bests in class for the measurement of some T1D autoantibodies. However, RBA is crucially dependent on operators’ expertise and poorly scalable in terms of throughput. Moreover, RBA is relatively costly, in part because of its low level of automation and the need to manage and dispose of radioactive substances. Several alternative immunoassays for T1D autoantibody measurement have been developed over the last 10 years that dispose of radioactive tracers. Only a few of these immunoassay formats have found routine application in T1D diagnostics or in pilot screening programs for T1D prediction. However, T1D autoantibody assays have not found wide implementation on high throughput Chemi Luminescent Immuno Assay (CLIA) platforms that are currently predominant in diagnostic laboratories.
Description of Project
Population-based screening programs for individuals at-risk of developing T1D are needed to offer therapeutic interventions aimed at delaying or forestalling overt diabetes. Currently, all strategies for the identification of persons at risk of T1D are crucially dependent on the measurement of multiple T1D autoantibodies. Current gold standard T1D autoantibody assays are based on formats which are incompatible with high throughput platforms commonly used in clinical diagnostics laboratories. With the support of JDRF and the Leona Helmsley foundation we previously developed novel recombinant autoantigens tagged with a luciferase reporter. We then successfully implemented their use for the measurement of all major T1D autoAbs in non-radioactive liquid phase Luciferase Immuno Precipitation System (LIPS) immunoassays. In this proposal, we plan to transfer the lessons learned in recombinant antigen design and assay set-up to a commercial Chemi Luminescent Immuno Assay (CLIA) platform widely adopted by diagnostic laboratories in >100 countries worldwide (LIAISON®, DiaSorin S.p.A., Italy).
Anticipated Outcome
If successful, our project would make T1D autoantibody testing easier and cheaper by leveraging the very large installed user base of CLIA immunodiagnostic platforms available worldwide. Furthermore, our project will generate reagents and know-how that might find applications also in other research and diagnostics settings.
Relevance to T1D
Population-based screening programs for individuals at-risk of developing T1D are needed to offer therapeutic interventions aimed at delaying or forestalling overt diabetes. The detection and quantification of T1D autoantibodies is the cornerstone of all current screening strategies. A successful implementation of T1D immunoassays on current high throughput CLIA diagnostics platforms would facilitate the introduction of such screening programs since it would leverage the vast installed base of CLIA instruments in diagnostics laboratories. Moreover, the adoption of equipment and reagents in common with other immune-enzymatic assays is expected to drive down assay costs in light of the associated major economies of scale.