Glucagon Quantikine ELISA Kit Summary

Product Summary

The Quantikine Glucagon Immunoassay is a 4.5 hour solid-phase ELISA designed to measure Glucagon in cell culture supernates, serum, and plasma. It contains natural porcine Glucagon as the standard. The antibodies were raised against a human Glucagon synthetic peptide. This immunoassay has been shown to accurately quantitate human, mouse, rat, and porcine Glucagon.

Preparation and Storage

Background: Glucagon

Glucagon is a 29 amino acid (aa) peptide produced by the pancreas that plays a critical role in glucose metabolism and homeostasis (1-4). The Glucagon precursor mRNA is expressed by alpha cells ( alpha -cells) of the pancreas, L cells of the intestine, and in the brain (1, 2). Only the pancreatic alpha -cells express the prohormone convertase PC2, also called PCSK2, which is required to produce Glucagon (2). Intestinal L cells instead express the prohormone convertase PC1, which processes the precursor to the Glucagon-overlapping peptides glicentin and oxyntomodulin. L cells also produce two Glucagon-like peptides, GLP-1 and GLP-2 that are derived from the same Glucagon precursor and influence glucose metabolism, but do not share any common sequence with Glucagon (1, 2). The aa sequence of the mature Glucagon peptide is identical in human, mouse, rat, pig, dog, horse, cow, sheep, and Xenopus. 

In normal metabolism, Glucagon is secreted in response to low blood glucose (hypoglycemia) and downregulated in response to high blood glucose (hyperglycemia). Although Glucagon binding sites are found in liver, brain, pancreas, kidney, intestine, and adipose tissue, the main activity of Glucagon receptors occurs in the liver, where Glucagon stimulates gluconeogenesis and glycogenolysis, thereby increasing blood glucose (1-4). It is particularly important that the brain receive sufficient glucose, since it is unable to store more than a minute quantity. Therefore the release of Glucagon from alpha -cells is under control by both hormones and neurotransmitters, and is very responsive to circulating glucose concentration. Insulin, and/or the zinc that islet beta cells secrete with it, downregulates Glucagon secretion in intact islets (5, 6). Glucagon secretion is also downregulated by the neurotransmitter gamma -aminobutyric acid (GABA), somatostatin produced by islet δ-cells, and GLP-1, but is enhanced by the neurotransmitter L-glutamate, amino acids (especially arginine), and Glucagon itself (2-4, 7). Through receptors on the alpha -cells, these substances affect potassium, sodium, and calcium channel activity and alter intracellular calcium concentration (2-4). Glucose suppression of Glucagon secretion is probably indirect, acting through paracrine signals from other islet cells (8). 

Like insulin, Glucagon is dysregulated in type 2 diabetes (T2D) and contributes to its pathology (2-4). Glucagon secretion is less responsive to insulin-mediated suppression in times of high circulating glucose, causing glucagonemia, and increasing the risk of hyperglycemia. Glucagon is also regulated by some of the same messengers that regulate insulin (10-12). Leptin inhibits alpha -cell glucagon secretion and stimulates beta -cell insulin secretion, but glucagon blunts the leptin-mediated insulin secretion (10). Islet alpha -cells express ghrelin receptors and respond to ghrelin by increasing Glucagon secretion (11). Glucocorticoids, activated by 11 beta -HSD1, depress Glucagon secretion in hypoglycemia and insulin secretion in hyperglycemia (12). Although genetic polymorphisms of the Glucagon receptor are associated with T2D, downregulation of Glucagon secretion or deletion of the Glucagon receptor in mice that are susceptible to T2D actually improves glycemic control (13, 14).

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1、Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2、Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3、Add 50 μL of Assay Diluent to each well.

4、Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5、Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6、Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7、Aspirate and wash 5 times.

8、Add 100 μL Substrate Solution to each well.

9、Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.