Description

Nori Equine Insulin ELISA Kit Summary

Alternative names for insulin: INS

Alternative names for equine: horse

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

Insulin is a peptide hormone produced by beta cells of the pancreatic islets and is the main anabolic hormone of the body. It regulates the metabolism of carbohydrates, fats and protein by promoting the absorption of glucose from the blood into liver, fat and skeletal muscle cells. In these tissues the absorbed glucose is converted into either glycogen via glycogenesis or fats (triglycerides) via lipogenesis, or, in the case of the liver, into both. Glucose production and secretion by the liver is strongly inhibited by high concentrations of insulin in the blood.^[1] Circulating insulin also affects the synthesis of proteins in a wide variety of tissues. It promotes the conversion of small molecules in the blood into large molecules inside the cells. Low insulin levels in the blood have the opposite effect by promoting widespread catabolism, especially of reserve body fat. Beta cells are sensitive to blood sugar levels so that they secrete insulin into the blood in response to high level of glucose; and inhibit secretion of insulin when glucose levels are low.^[2] Insulin enhances glucose uptake and metabolism in the cells, thereby reducing blood sugar level. The secretion of insulin and glucagon into the blood in response to the blood glucose concentration is the primary mechanism of glucose homeostasis.^[2] Decreased or absent insulin activity results in diabetes mellitus, a condition of high blood sugar level (hyperglycaemia). There are two types of the disease. In type 1 diabetes mellitus, the beta cells are destroyed by an autoimmune reaction so that insulin can no longer be synthesized or be secreted into the blood. In type 2 diabetes mellitus, the destruction of beta cells is less pronounced than in type 1 diabetes, and is not due to an autoimmune process. Instead, there is an accumulation of amyloid in the pancreatic islets, which likely disrupts their anatomy and physiology.^[2] The pathogenesis of type 2 diabetes is not well understood but reduced population of islet beta-cells, reduced secretory function of islet beta-cells that survive, and peripheral tissue insulin resistance are known to be involved. Type 2 diabetes is characterized by increased glucagon secretion which is unaffected by, and unresponsive to the concentration of blood glucose. But insulin is still secreted into the blood in response to the blood glucose.^[2] As a result, glucose accumulates in the blood.

References

1. Sonksen P, Sonksen J (2000). British Journal of Anaesthesia. 85(1): 69–79.
2. Koeslag JH, et al. (2003). The Journal of Physiology. 549 (Pt 2): 333–46.

Product Citations