Daniela Liberati1,2, Rebecca C Wyatt3, Cristina Brigatti2, Ilaria Marzinotto2, Maurizio Ferrari1,4, Elena Bazzigaluppi5, Emanuele Bosi2,4, Ben T Gillard3, Kathleen M Gillespie3, Frans Gorus6,7, Ilse Weets6,7, Eric Balti6,7, Lorenzo Piemonti2,4, Peter Achenbach8, Alistair J K Williams3, Vito Lampasona9. 1. Human Pathology Genomic Diagnostics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Via Olgettina 60, 20132, Milan, Italy. 2. Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Milan, Italy. 3. Diabetes and Metabolism Unit, Translational Health Sciences, University of Bristol, Bristol, UK. 4. Vita-Salute San Raffaele University, Milan, Italy. 5. Department of Laboratory Medicine, IRCCS San Raffaele Scientific Institute, Milan, Italy. 6. Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium. 7. Department of Clinical Chemistry, Universitair Ziekenhuis Brussel, Brussels, Belgium. 8. Institute of Diabetes Research, Helmholtz Zentrum München, and Forschergruppe Diabetes, Klinikum rechts der Isar, Technische Universität München, Neuherberg, Germany. 9. Human Pathology Genomic Diagnostics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Via Olgettina 60, 20132, Milan, Italy. lampasona.vito@hsr.it.
Abstract
AIMS: Insulin autoantibodies (IAA) are often the first marker of autoimmunity detected in children in the preclinical phase of type 1 diabetes (T1D). Currently, the vast majority of laboratories adopt the radiobinding micro-assay (RBA) for measuring IAA. Our aim was to replace RBA with a novel non-radioactive IAA Luciferase Immuno Precipitation System (LIPS) assay with improved performance. METHODS: We developed (pro)insulin antigens with alternative placements of a NanoLuc™ luciferase reporter (NLuc). Performance in LIPS was evaluated by testing sera from new onset T1D (n = 80), blood donors (n = 123), schoolchildren (n = 186), first-degree relatives (FDRs) from the Bart's Oxford family study (n = 53) and from the Belgian Diabetes Registry (n = 136), coded sera from the Islet Autoantibody Standardization Program (IASP) (T1D n = 50, blood donors n = 90). RESULTS: IAA LIPS based on B chain-NLuc proinsulin or B chain-NLuc insulin, in which NLuc was fused at the C-terminus of the insulin B chain, required only 2 μL of serum and a short incubation time, showed high concordance with RBA (Spearman r = 0.866 and 0.833, respectively), high assay performance (B chain-NLuc proinsulin ROC-AUC = 0.894 and B chain-NLuc insulin ROC-AUC = 0.916), and an adjusted sensitivity at 95% specificity ranking on par with the best assays submitted to the two most recent IASP workshops. In FDRs, the IAA LIPS showed improved discrimination of progressors to T1D compared to RBA. CONCLUSIONS: We established a novel high-performance non-radioactive IAA LIPS that might replace the current gold standard RBA and find wide application in the study of the IAA response in T1D.
AIMS: Insulin autoantibodies (IAA) are often the first marker of autoimmunity detected in children in the preclinical phase of type 1 diabetes (T1D). Currently, the vast majority of laboratories adopt the radiobinding micro-assay (RBA) for measuring IAA. Our aim was to replace RBA with a novel non-radioactive IAA Luciferase Immuno Precipitation System (LIPS) assay with improved performance. METHODS: We developed (pro)insulin antigens with alternative placements of a NanoLuc™ luciferase reporter (NLuc). Performance in LIPS was evaluated by testing sera from new onset T1D (n = 80), blood donors (n = 123), schoolchildren (n = 186), first-degree relatives (FDRs) from the Bart's Oxford family study (n = 53) and from the Belgian Diabetes Registry (n = 136), coded sera from the Islet Autoantibody Standardization Program (IASP) (T1D n = 50, blood donors n = 90). RESULTS:IAA LIPS based on B chain-NLuc proinsulin or B chain-NLuc insulin, in which NLuc was fused at the C-terminus of the insulin B chain, required only 2 μL of serum and a short incubation time, showed high concordance with RBA (Spearman r = 0.866 and 0.833, respectively), high assay performance (B chain-NLuc proinsulin ROC-AUC = 0.894 and B chain-NLuc insulin ROC-AUC = 0.916), and an adjusted sensitivity at 95% specificity ranking on par with the best assays submitted to the two most recent IASP workshops. In FDRs, the IAA LIPS showed improved discrimination of progressors to T1D compared to RBA. CONCLUSIONS: We established a novel high-performance non-radioactive IAA LIPS that might replace the current gold standard RBA and find wide application in the study of the IAA response in T1D.
Authors: Kenney Ng; Vibha Anand; Harry Stavropoulos; Riitta Veijola; Jorma Toppari; Marlena Maziarz; Markus Lundgren; Kathy Waugh; Brigitte I Frohnert; Frank Martin; Olivia Lou; William Hagopian; Peter Achenbach Journal: Diabetologia Date: 2022-10-05 Impact factor: 10.460
Authors: Kenney Ng; Harry Stavropoulos; Vibha Anand; Riitta Veijola; Jorma Toppari; Marlena Maziarz; Markus Lundgren; Kathy Waugh; Brigitte I Frohnert; Frank Martin; William Hagopian; Peter Achenbach Journal: Diabetes Care Date: 2022-01-01 Impact factor: 17.152
Authors: Markus Hippich; Lisa Holthaus; Robin Assfalg; Jose Zapardiel-Gonzalo; Heidi Kapfelsperger; Martin Heigermoser; Florian Haupt; Dominik A Ewald; Tiziana C Welzhofer; Benjamin A Marcus; Susanne Heck; Annika Koelln; Joanna Stock; Franziska Voss; Massimiliano Secchi; Lorenzo Piemonti; Kathrin de la Rosa; Ulrike Protzer; Merle Boehmer; Peter Achenbach; Vito Lampasona; Ezio Bonifacio; Anette-Gabriele Ziegler Journal: Med (N Y) Date: 2020-10-29