Simultaneous measurement of plasma concentrations of proinsulin and C-peptide and their ratio with a trefoil-type time-resolved fluorescence immunoassay.
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.
De Pauw
Pieter E M
PE
Diabetes Research Center, Brussels Free University, Brussels, Belgium.
Vermeulen
Ilse
I
Ubani
Ogonnaya C
OC
Truyen
Inge
I
Vekens
Evilien M F
EM
van Genderen
Farah T
FT
De Grijse
Joeri W
JW
Pipeleers
Daniel G
DG
Van Schravendijk
Chris
C
Gorus
Frans K
FK
D000911:Antibodies, Monoclonal; D001323:Autoantibodies / Q000097:blood; D002096:C-Peptide / Q000097:blood* / Q000276:immunology; D003922:Diabetes Mellitus, Type 1 / Q000175:diagnosis / Q000276:immunology; D005455:Fluorescent Antibody Technique; D006801:Humans; D011384:Proinsulin / Q000097:blood* / Q000276:immunology
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D002096:C-Peptide; D011384:Proinsulin