A time-resolved fluorescence immunoassay for insulin in rodent plasma

Abstract

We describe a time-resolved fluoroimmunoassay (TR-FIA) for quantification of insulin in rodent serum and plasma in the picomolar levels typical of these samples. The method is a solid-phase, sequential saturation assay based on competition of unlabeled insulin and biotinamidocaproyl-labeled insulin for anti-insulin antibody. Europium-labeled streptavidin allows the DELFIA system (Wallac) to be used for detection. The assay is sensitive (0.1 fmol detection limit, EC 50=58±3 pM), accurate (>95% recovery of 88–880 pM insulin added to the samples), and simple enough to be automated in a 96-well microtiter plate format. Blood samples of 5 μl can be quickly processed and analyzed within a working concentration range of 40–200 pM, allowing direct measurement of insulin levels in rodents from a tail bleed. We used the TR-FIA to assess insulin levels in mouse and rat samples. In studies of streptozotocin-induced diabetes, as well as glucose load experiments, the assay gave results consistent with known literature. The measured insulin levels correlated significantly with values obtained by radioimmunoassay ( R 2=0.996). The intra-assay and inter-assay coefficients of variation were 2.3% and 15%, respectively. We compared results of this assay with an enzyme-linked immunosorbent assay (ELISA) method. The TR-FIA method was comparable to the ELISA but had higher sensitivity and required only one-tenth as much sample. The assay can be performed using commercially available reagents that allow for high sensitivity and practicability.