A time-resolved fluorescence immunoassay for insulin in rodent plasma.
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.
Daijo
J E
JE
Telik, Incorporated, South San Francisco, CA 94080, USA.
Sportsman
J R
JR
D000818:Animals; D001786:Blood Glucose; D003921:Diabetes Mellitus, Experimental / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay; D005069:Evaluation Studies as Topic; D015200:Fluoroimmunoassay / Q000379:methods* / Q000592:standards; D005951:Glucose Tolerance Test; D007328:Insulin / Q000097:blood*; D051379:Mice; D008810:Mice, Inbred C57BL; D008820:Mice, Obese; D011863:Radioimmunoassay; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001786:Blood Glucose; D007328:Insulin