Canine IL-6 Quantikine Kit Summary

Product Summary

The Quantikine Canine IL-6 Immunoassay is a 4.5 hour solid-phase ELISA designed to measure canine IL-6 in cell culture supernates, serum, and plasma. It contains E. coli-expressed recombinant canine IL-6 and antibodies raised against the recombinant factor. This immunoassay has been shown to accurately quantitate the recombinant factor. Results obtained using natural canine IL-6 showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for naturally occurring canine IL-6.

Preparation and Storage

Background: IL-6

Interleukin 6 (IL-6) is a pleiotropic, alpha -helical, 22-28 kDa phosphorylated and variably glycosylated cytokine that plays important roles in the acute phase reaction, inflammation, hematopoiesis, bone metabolism, and cancer progression (1-5). Mature canine IL-6 is 187 amino acids (aa) in length and shares 58%, 37%, 39%, and 76% aa sequence identity with human, mouse, rat, and feline IL-6, respectively. Cells known to express IL-6 include CD8+ T cells, fibroblasts, synoviocytes, adipocytes, osteoblasts, megakaryocytes, endothelial cells (under the influence of endothelins), sympathetic neurons, cerebral cortex neurons, adrenal medulla chromaffin cells, retinal pigment cells, mast cells, keratinocytes, Langerhans cells, fetal and adult astrocytes, neutrophils, monocytes, eosinophils, colonic epithelial cells, B1 B cells and pancreatic islet beta cells (2, 6-28). IL-6 production is generally correlated with cell activation and is normally kept in control by glucocorticoids, catecholamines, and secondary sex steroids (2). Normal human circulating IL-6 is in the 1 pg/mL range, with slight elevations during the menstrual cycle, modest elevations in certain cancers, and large elevations after surgery (29-33). 

IL-6 induces signaling through a cell surface heterodimeric receptor complex composed of a ligand binding subunit (IL-6 R alpha) and a signal transducing subunit (gp130). IL-6 binds to IL-6 R alpha, triggering IL-6 R alpha association with gp130 and gp130 dimerization (34). gp130 is also a component of the receptors for CLC, CNTF, CT-1, IL-11, IL-27, LIF, and OSM (35). Soluble forms of IL-6 R alpha are generated by both alternative splicing and proteolytic cleavage (5). In a mechanism known as trans-signaling, complexes of soluble IL-6 and IL-6 R alpha elicit responses from gp130- expressing cells that lack cell surface IL-6 R alpha (5). Trans-signaling enables a wider range of cell types to respond to IL-6, as the expression of gp130 is ubiquitous, while that of IL-6 R alpha is predominantly restricted to hepatocytes, monocytes, and resting lymphocytes (2, 5). Soluble splice forms of gp130 block trans-signaling from IL-6/IL-6 R alpha but not from other cytokines that use gp130 as a co-receptor (5, 36). 

IL-6, along with TNF-alpha and IL-1, drives the acute inflammatory response. IL-6 is almost solely responsible for fever and the acute phase response in the liver, and it is important in the transition from acute inflammation to either acquired immunity or chronic inflammatory disease (1-5). When dysregulated, it contributes to chronic inflammation in conditions such as obesity, insulin resistance, inflammatory bowel disease, arthritis, and sepsis (2, 5). IL-6 modulates bone resorption and is a major effector of inflammatory joint destruction in rheumatoid arthritis through its promotion of Th17 cell development and activity (1). It contributes to atherosclerotic plaque development and destabilization as well as the development of inflammation-associated carcinogenesis (1, 2). IL-6 can also function as an anti-inflammatory molecule, as in skeletal muscle where it is secreted in response to exercise (2). In addition, it enhances hematopoietic stem cell proliferation and the differentiation of memory B cells and plasma cells (37).

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.