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- Assay Type Sandwich ELISA
- Sample Types Serum, Plasma, Tissue Culture Media
- Sensitivity 48.8 pg/mL
- Species Human, Bovine, Porcine
- Assay Duration 2 Hours
- Samples/Plate 42 in Duplicate
- Readout Colorimetric, 450 nm
- Standard Curve
An Insulin standard, calibrated to the WHO International Standard 1975 (66/304), is provided to generate a standard curve for the assay and all samples should be read off the standard curve. Standards or diluted samples are pipetted into a clear microtiter plate coated with a monoclonal antibody to capture insulin present in the sample. After a 60 minute incubation, the plate is washed. A peroxidase conjugated insulin antibody is added and the plate is again incubated for 30 minutes and washed. Substrate is then added to the plate, which reacts with the bound insulin conjugated antibody. After a third incubation, the reaction is stopped and the intensity of the generated color is read at 450 nm.
The human insulin protein is a 51 amino acid anabolic peptide-hormone that is secreted by the pancreatic β-cells in the Islets of Langerhans. Insulin consists of two chains (A and B) connected by disulfide bonds. One of its primary functions is the stimulation of glucose uptake from the systemic circulation, as well as the suppression of hepatic gluconeogenesis, thereby serving a major role in glucose homeostasis and prevention of the metabolic disorder diabetes mellitus.
The work of Banting, Best, Collip and MacCleod in the early 1920’s resulted in the identification of a substance in extracts of pancreas that had the remarkable ability to reduce blood glucose levels in diabetic animals. By 1923, these pancreatic extracts were being used to successfully treat diabetic patients. Insulin exists primarily as a monomer at low concentrations(~10-6 M) and forms dimers at higher concentrations and neutral pH. At high concentrations and in the presence of zinc ions insulin aggregates further to form hexameric complexes. Preproinsulin, the first translational product from the insulin gene, is a 110 amino acid polypeptide with a 24 amino acid signal peptide.
The major function of insulin is to counter the concerted actions of a number of hyperglycemia-generating hormones and to maintain low blood glucose levels. In addition to its role in regulating glucose metabolism, insulin stimulates lipogenesis, diminishes lipolysis, and increases amino acid transport into cells. Because there are numerous hyperglycemic hormones, untreated disorders associated with insulin generally lead to severe hyperglycemia and shortened life span.