Rat IL-6 Quantikine ELISA Kit Summary

Product Summary

The Quantikine Rat IL-6 Immunoassay is a 4.5 hour solid phase ELISA designed to measure rat IL-6 in cell culture supernates, rat serum, and plasma. It contains E. coli-expressed recombinant rat IL-6 and antibodies raised against the recombinant factor. This immunoassay has been shown to accurately quantitate the recombinant protein. Results obtained using natural rat IL-6 showed dose-response 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 natural rat IL-6.

Preparation and Storage

Background: IL-6

Interleukin 6 (IL-6) is a multifunctional cytokine that plays important roles in host defense, acute phase reactions, immune responses, nerve cell functions and hematopoiesis (1-5). It is expressed by a variety of normal and transformed lymphoid and non-lymphoid cells. The production of IL-6 is up-regulated by numerous signals such as mitogenic or antigenic stimulation, lipopolysaccharides, calcium ionophores, cytokines and viruses. IL-4, IL-10 and IL-13 inhibit IL-6 expression in monocytes. Elevated serum IL-6 levels have been observed in a number of pathological conditions, including bacterial and viral infections, trauma, autoimmune diseases, inflammations and malignancies (1-5). 

IL-6 is a prototypic member of the IL-6 superfamily of cytokines that share gp130 as a component required for signal transduction (4). The rat, mouse and human IL-6 cDNAs have been cloned (6-9). Rat IL-6 cDNA encodes a 211 amino acid (aa) residue precursor polypeptide with a hydrophobic signal peptide that is cleaved to generate the 187 aa residue mature protein. At the protein sequence level, there is approximately 39% identity between rat and human, and 87% identity between mouse and rat IL-6 (6). Although human and mouse IL-6 are equally active on mouse cells, mouse IL-6 is not active on human cells (9). 

The high-affinity IL-6 receptor complex, which mediates IL-6 bioactivity, consists of two membrane glycoproteins: an 80 kDa low-affinity IL-6-binding receptor (IL-6 R) and a 130 kDa signal-transducing protein (gp130) that lacks IL-6 binding ability (4). The binding of IL-6 to IL-6 R recruits gp130 to form a trimeric complex that dimerizes to form a hexameric complex, which transduces IL-6 signal (4). Soluble forms of both IL-6 R and gp130 have been detected in blood (11, 12). Soluble IL-6 R is capable of binding IL-6 and inducing homodimerization of membrane gp130 and subsequent signal transduction.

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.