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.
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.
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.
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.
We present a time-resolved fluorescence immunoassay (TR–FIA) for the measurement of rat insulin in cell extracts and culture media. This assay is based on the binding of two monoclonal antibodies to ...different parts of the insulin molecule in a 96-well microtiter plate. For the detection, europium-labeled streptavidin that interacts with the second biotinylated antibody is used. Samples of 25
μl could be analyzed in less than 2
days with a measuring range between 5 and 1250
pg (0.2–50
μg/L or 34.4–8600
pM). The inter- and intraassay percentage coefficients of variation were less than 8.3 and 5.1, respectively. Recoveries of 0.48 to 40
μg/L rat insulin, added to culture medium, ranged between 94 and 107%. Results were significantly correlated with those of an in-house radioimmunoassay (RIA) for rodent insulin (
P
<
0.0001,
r
2
=
0.99). The TR–FIA method had a similar detection limit (0.16
μg/L), but its working range was at least 5-fold larger. Additional advantages include the lower cost, the applicability to measurements in tissue and serum, and the quantification of insulin from other species.
