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Clinical Chemistry 54: 1990-1998, 2008. First published October 9, 2008; 10.1373/clinchem.2008.109710
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(Clinical Chemistry. 2008;54:1990-1998.)
© 2008 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

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 Pauw1,2, Ilse Vermeulen1,2, Ogonnaya C. Ubani1, Inge Truyen1, Evilien M.F. Vekens1, Farah T. van Genderen1, Joeri W. De Grijse1, Daniel G. Pipeleers1, Chris Van Schravendijk1 and Frans K. Gorus1,a

1 Diabetes Research Center, Brussels Free University, Brussels, Belgium.

aAddress correspondence to this author at: Department of Clinical Chemistry and Radioimmunology, Brussels University Hospital (UZ-Brussel), 101 Laarbeeklaan, B-1090 Brussels, Belgium. Fax +32 2 477 50 60; e-mail Frans.Gorus{at}uzbrussel.be.

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 (r2 ≥ 0.965; P < 0.0001). The total error for PI/C obtained with the trefoil-type TRFIA remained ≤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.






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