title: Development of an Enhanced Sensitivity Bead-Based Immunoassay for Real-Time In Vivo Detection of Pancreatic β-Cell Death.
abstract: There is a clinical need for plasma tests to detect and quantify the in vivo destruction of pancreatic β-cells in type 1 diabetes. We previously developed a time-resolved fluorescence immunoassay (TRFIA) to glutamate decarboxylase 65 kDa (GAD65) (GAD65-TRFIA) that was able to detect the synchronous necrotic destruction of transplanted β-cells in the hours after their infusion in the liver. This GAD65-TRFIA, however, lacked sensitivity to detect continued β-cell rejection beyond this acute phase. The aim of present study was to gain at least an order of magnitude in analytical sensitivity by switching to Becton Dickinson cytometric bead array (CBA) (GAD65-CBA) enhanced sensitivity format, using the same couple of monoclonal antibodies. We compared the performances of GAD65-CBA and GAD65-TRFIA using Clinical and Laboratory Standards Institute protocols for linearity, imprecision, specificity, limit of detection, and functional sensitivity. We conducted a method comparison and assessed the biologic potential on samples from human recipients of islet grafts. The GAD65-CBA showed acceptable linearity and imprecision. Switching from TRFIA to CBA lowered functional sensitivity by a factor 35 and lowered limit of detection by a factor 11 with minimal need for method optimization. The enhanced sensitivity greatly expands the application domain of our biomarker and allowed for the first time to detect ongoing β-cell destruction up to at least 1 day after islet transplantation. We conclude that the GAD65-CBA is suitable for biological and clinical assessment of the real-time destruction of β-cells in intraportal transplantation.
authors.affiliation: Diabetes Research Center (O.C., G.S., B.B., E.N., Z.L., D.P., F.K.G., G.A.M.), Brussels Free University and Department of Clinical Chemistry and Radio-Immunology (O.C., K.V., B.B., F.K.G., G.A.M.), Universitair Ziekenhuis Brussel, B1090 Brussels, Belgium; and Department of Medicine (C.S.H.), University of Washington, Seattle, Washington 98109-4725.
authors.forename: Olivier R
authors.initials: OR
authors.lastname: Costa
authors.affiliation: Diabetes Research Center (O.C., G.S., B.B., E.N., Z.L., D.P., F.K.G., G.A.M.), Brussels Free University and Department of Clinical Chemistry and Radio-Immunology (O.C., K.V., B.B., F.K.G., G.A.M.), Universitair Ziekenhuis Brussel, B1090 Brussels, Belgium; and Department of Medicine (C.S.H.), University of Washington, Seattle, Washington 98109-4725.
authors.forename: Geert
authors.initials: G
authors.lastname: Stangé
authors.affiliation: Diabetes Research Center (O.C., G.S., B.B., E.N., Z.L., D.P., F.K.G., G.A.M.), Brussels Free University and Department of Clinical Chemistry and Radio-Immunology (O.C., K.V., B.B., F.K.G., G.A.M.), Universitair Ziekenhuis Brussel, B1090 Brussels, Belgium; and Department of Medicine (C.S.H.), University of Washington, Seattle, Washington 98109-4725.
authors.forename: Katrijn
authors.initials: K
authors.lastname: Verhaeghen
authors.affiliation: Diabetes Research Center (O.C., G.S., B.B., E.N., Z.L., D.P., F.K.G., G.A.M.), Brussels Free University and Department of Clinical Chemistry and Radio-Immunology (O.C., K.V., B.B., F.K.G., G.A.M.), Universitair Ziekenhuis Brussel, B1090 Brussels, Belgium; and Department of Medicine (C.S.H.), University of Washington, Seattle, Washington 98109-4725.
authors.forename: Benedicte
authors.initials: B
authors.lastname: Brackeva
authors.affiliation: Diabetes Research Center (O.C., G.S., B.B., E.N., Z.L., D.P., F.K.G., G.A.M.), Brussels Free University and Department of Clinical Chemistry and Radio-Immunology (O.C., K.V., B.B., F.K.G., G.A.M.), Universitair Ziekenhuis Brussel, B1090 Brussels, Belgium; and Department of Medicine (C.S.H.), University of Washington, Seattle, Washington 98109-4725.
authors.forename: Ellen
authors.initials: E
authors.lastname: Nonneman
authors.affiliation: Diabetes Research Center (O.C., G.S., B.B., E.N., Z.L., D.P., F.K.G., G.A.M.), Brussels Free University and Department of Clinical Chemistry and Radio-Immunology (O.C., K.V., B.B., F.K.G., G.A.M.), Universitair Ziekenhuis Brussel, B1090 Brussels, Belgium; and Department of Medicine (C.S.H.), University of Washington, Seattle, Washington 98109-4725.
authors.forename: Christiane S
authors.initials: CS
authors.lastname: Hampe
authors.affiliation: Diabetes Research Center (O.C., G.S., B.B., E.N., Z.L., D.P., F.K.G., G.A.M.), Brussels Free University and Department of Clinical Chemistry and Radio-Immunology (O.C., K.V., B.B., F.K.G., G.A.M.), Universitair Ziekenhuis Brussel, B1090 Brussels, Belgium; and Department of Medicine (C.S.H.), University of Washington, Seattle, Washington 98109-4725.
authors.forename: Zhidong
authors.initials: Z
authors.lastname: Ling
authors.affiliation: Diabetes Research Center (O.C., G.S., B.B., E.N., Z.L., D.P., F.K.G., G.A.M.), Brussels Free University and Department of Clinical Chemistry and Radio-Immunology (O.C., K.V., B.B., F.K.G., G.A.M.), Universitair Ziekenhuis Brussel, B1090 Brussels, Belgium; and Department of Medicine (C.S.H.), University of Washington, Seattle, Washington 98109-4725.
authors.forename: Daniel
authors.initials: D
authors.lastname: Pipeleers
authors.affiliation: Diabetes Research Center (O.C., G.S., B.B., E.N., Z.L., D.P., F.K.G., G.A.M.), Brussels Free University and Department of Clinical Chemistry and Radio-Immunology (O.C., K.V., B.B., F.K.G., G.A.M.), Universitair Ziekenhuis Brussel, B1090 Brussels, Belgium; and Department of Medicine (C.S.H.), University of Washington, Seattle, Washington 98109-4725.
authors.forename: Frans K
authors.initials: FK
authors.lastname: Gorus
authors.affiliation: Diabetes Research Center (O.C., G.S., B.B., E.N., Z.L., D.P., F.K.G., G.A.M.), Brussels Free University and Department of Clinical Chemistry and Radio-Immunology (O.C., K.V., B.B., F.K.G., G.A.M.), Universitair Ziekenhuis Brussel, B1090 Brussels, Belgium; and Department of Medicine (C.S.H.), University of Washington, Seattle, Washington 98109-4725.
authors.forename: Geert A
authors.initials: GA
authors.lastname: Martens
mesh_terms: D017209:Apoptosis; D015415:Biomarkers / Q000097:blood; D003922:Diabetes Mellitus, Type 1 / Q000097:blood* / Q000628:therapy; D015200:Fluoroimmunoassay / Q000379:methods; D005968:Glutamate Decarboxylase / Q000097:blood*; D006801:Humans; D007118:Immunoassay / Q000379:methods; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D016381:Islets of Langerhans Transplantation; D008863:Microspheres
chemical_list: D015415:Biomarkers; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2