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Updated: Aug 8, 2026
Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion
Published on: May 10, 2018
A time-resolved fluorescence immunoassay for insulin in rodent plasma
1Telik, Incorporated, South San Francisco, CA 94080, USA.
Insights
A new time-resolved fluoroimmunoassay (TR-FIA) accurately quantifies rodent insulin levels in picomolar ranges. This sensitive and practical assay requires minimal sample volume, enabling precise insulin measurement in mice and rats.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Endocrinology
Background:
- Accurate insulin quantification is crucial for rodent diabetes and metabolic research.
- Existing methods may lack sensitivity or require large sample volumes.
- Picomolar insulin levels in rodents necessitate highly sensitive assays.
Purpose of the Study:
- To develop and validate a time-resolved fluoroimmunoassay (TR-FIA) for precise insulin measurement in rodent serum and plasma.
- To establish an assay with high sensitivity, accuracy, and minimal sample volume requirements.
- To enable direct insulin level assessment in small rodent blood samples.
Main Methods:
- Solid-phase, sequential saturation assay using biotinamidocaproyl-labeled insulin.
- Competition assay with unlabeled insulin and labeled insulin for anti-insulin antibody.
- Detection via Europium-labeled streptavidin and the DELFIA system.
Main Results:
- Sensitive assay with a 0.1 fmol detection limit and EC50 of 58 +/- 3 pM.
- Accurate quantification with >95% recovery for added insulin (88-880 pM).
- Assay validated against radioimmunoassay (R2 = 0.996) and ELISA, showing higher sensitivity and lower sample requirement (1/10th).
Conclusions:
- The TR-FIA is a sensitive, accurate, and practical method for quantifying rodent insulin.
- The assay is suitable for automation and analysis of small sample volumes (5 microl) from tail bleeds.
- TR-FIA provides reliable insulin measurements comparable to established methods but with enhanced sensitivity and efficiency.
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, EC50 = 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 microl 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 (R2 = 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.
