Simultaneous measurement of plasma concentrations of proinsulin and C-peptide and their ratio with a trefoil-type time-resolved fluorescence immunoassay

Pieter E M De Pauw, Ilse Vermeulen, Ogonnaya C Ubani, Inge Truyen, Evilien M F Vekens, Farah T van Genderen, Joeri W De Grijse, Daniel G Pipeleers, Chris Van Schravendijk, Frans K Gorus

Research output: Contribution to journalArticlepeer-review

Abstract

BACKGROUND: When the concentrations of 2 or more substances are measured separately, their molar ratios are subject to the additive imprecisions of the different assays. We hypothesized that the cumulative error for concentration ratios of peptides containing a common sequence might be minimized by measuring the peptides simultaneously with a "trefoil-type" immunoassay.

METHODS: As a model of this approach, we developed a dual-label time-resolved fluorescence immunoassay (TRFIA) to simultaneously measure proinsulin, C-peptide, and the proinsulin-C-peptide ratio (PI/C). A monoclonal antibody captures all C-peptide-containing molecules, and 2 differently labeled antibodies distinguish between proinsulin-like molecules and true C-peptide.

RESULTS: The trefoil-type TRFIA was capable of measuring plasma C-peptide and proinsulin simultaneously without mutual interference at limits of quantification of 48 and 8125 pmol/L, and 2.1 and 197 pmol/L, respectively. Within-laboratory imprecision values for the trefoil-type TRFIA ranged between 8.4% and 12% for the hormone concentrations. Unlike the hormone results obtained with separate assays, imprecision did not increase when PI/C was calculated from trefoil assay results (P < 0.05). Peptide concentrations were highly correlated with results obtained in individual comparison assays (r(2) > or = 0.965; P < 0.0001). The total error for PI/C obtained with the trefoil-type TRFIA remained < or = 25% over a broader C-peptide range than with separate hormone assays (79-7200 pmol/L vs 590-4300 pmol/L C-peptide). Preliminary data indicate little or no interference by heterophile antibodies.

CONCLUSIONS: The developed trefoil-type TRFIA is a reliable method for simultaneous measurement of proinsulin, C-peptide, and PI/C and provides proof of principle for the development of other trefoil-type multiple-label immunoassays.

Original languageEnglish
Pages (from-to)1990-1998
Number of pages9
JournalClinical Chemistry
Volume54
Issue number12
DOIs
Publication statusPublished - Dec 2008

Keywords

  • Antibodies, Monoclonal
  • Autoantibodies/blood
  • C-Peptide/blood
  • Diabetes Mellitus, Type 1/diagnosis
  • Fluorescent Antibody Technique
  • Humans
  • Proinsulin/blood

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