title: Sensitive and specific time-resolved fluorescence immunoassay of rat C-peptide for measuring hormone secretory and storage capacity of β-cells in vivo and in vitro.
abstract: The limitations of current rat C-peptide assays led us to develop a time-resolved fluorescence immunoassay for measurements in plasma, incubation media, and tissue/cell extracts. The assay uses 2 monoclonal antibodies, binding to different parts of the C-peptide molecule, and allowing, respectively, capture of the peptide and its detection by europium-labeled streptavidin. It is performed on 25-μL samples for a dynamic range from 66pM up to 3900pM C-peptide and displays over 95% recovery of added peptide in the range of 111pM to 2786pM. Its inter- and intra-assay coefficients of variations are, respectively, lower than 7.6% and 4.8%. Cross-reactivities by rat insulin and by human and porcine C-peptide are negligible, and cross-reactivity by mouse C-peptide is 6% ± 2%. The assay has been validated for in vivo and in vitro measurements of C-peptide release and cellular content. Release patterns were similar to those for insulin and occurred in equimolar concentrations for both peptides. The molar C-peptide contents in purified β-cells and isolated islets were similar to the corresponding insulin contents. This was also the case for pancreatic extracts containing protease inhibitors.
authors.affiliation: Department of Clinical Biology of Diabetes, Brussels University Hospital (UZ-Brussel), 101 Laarbeeklaan, B-1090 Brussels, Belgium. fvgender@vub.ac.be
authors.forename: Farah T
authors.initials: FT
authors.lastname: van Genderen
authors.forename: Frans K
authors.initials: FK
authors.lastname: Gorus
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Pipeleers
authors.forename: Christiaan F H
authors.initials: CF
authors.lastname: van Schravendijk
mesh_terms: D000818:Animals; D002096:C-Peptide / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D005453:Fluorescence; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D050417:Insulin-Secreting Cells / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D051381:Rats; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013552:Swine
chemical_list: D002096:C-Peptide