Human IL-6R alpha Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human IL-6 sR Immunoassay is a 4.5 hour solid phase ELISA designed to measure human IL-6 sR in cell culture supernates, serum, plasma, and urine. It contains recombinant human IL-6 sR produced in Sf 21 cells and antibodies raised against this protein. This kit has been shown to accurately quantitate the recombinant soluble IL-6 R. Results obtained using natural IL-6 sR 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 natural IL-6 sR. Since the measurement of human IL-6 sR by this immunoassay is insensitive to added recombinant human IL-6, it is probable that this measurement corresponds to the total amount of the soluble receptor present in samples ( i.e. the total amount of free receptor plus the total amount of receptor bound to IL-6).

Preparation and Storage

Background: IL-6R alpha

IL-6 R alpha is a transmembrane subunit of the receptor for Interleukin-6. It associates with gp130 which is a shared component of the receptors for CLC, CNTF, CT-1, IL-11, IL-27, LIF, and OSM. Soluble forms of IL-6 R alpha are generated by both alternative splicing and proteolytic cleavage. 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. IL-6 plays important roles in the acute phase reaction, inflammation, hematopoiesis, bone metabolism, and cancer progression. IL-6, along with TNF-alpha and IL-1, drives the acute inflammatory response and the transition from acute inflammation to either acquired immunity or chronic inflammatory disease. When dysregulated, it contributes to chronic inflammation in obesity, insulin resistance, inflammatory bowel disease, arthritis, sepsis, and atherosclerosis.

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.