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-L 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 interand intra-assay coefficients of variations are, respectively, lower than 7.6% and 4.8%. Crossreactivities by rat insulin and by human and porcine C-peptide are negligible, and cross-reactivity by mouse C-peptide is 6% Ϯ 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 ␤-cells and isolated islets were similar to the corresponding insulin contents. This was also the case for pancreatic extracts containing protease inhibitors. (Endocrinology 154: 1934 -1939, 2013)

Determination of Anticyclic Citrullinated Peptide Based on Biotin–Streptavidin‐Amplified Time‐Resolved Fluoroimmunoassay

“…In Figure 1, TRFIA demonstrated a good linear range within 9,463-0.58 U/ml, by contrast, within 591.4-18.48 U/ml of an ELISA kit. The expanded detection range will improve earlier detection and higher sensitivity in RA diagnosis (10,11). Furthermore, the serologic-positive patients will be treated timely and effectively, subsequently, preventing irreversible joint damage (12).…”

Control of (pre)-analytical aspects in immunoassay measurements of metabolic hormones in rodents

“…Important steps of this basic validation include the analysis of precision and linearity; the latter can for example be done with serial dilutions of the calibrators or positive controls, which should always be delivered with an immunoassay kit. Other recent examples of hormone-specific and extended, state-of-the-art assay validation efforts for novel immunoassays measuring hormones in samples from laboratory animal species can be found in the cited references ( 70 , 71 , 72 , 73 , 74 ). In addition, literature research or personal exchange during scientific meetings can be helpful to elucidate if other research groups have successfully used the (potentially novel) immunoassay to measure the analyte.…”