Human IL-1ra/IL-1F3 Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human IL-1ra Immunoassay is a 4.5 hour solid phase ELISA designed to measure IL-1ra in cell culture supernates, serum, and plasma. It contains E. coli-derived recombinant human IL-1ra as well as antibodies raised against the recombinant factor. This immunoassay has been shown to accurately quantitate recombinant human IL-1ra. Results obtained using natural human IL-1ra 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 human IL-1ra.

Preparation and Storage

Background: IL-1ra/IL-1F3

Interleukin-1 receptor antagonist (IL-1ra; also known as IL-1F3) is a 22-25 kDa member of the IL-1 family of cytokines. Currently, there are 11 family members (IL-1F1-F11), nine of which form an IL-1 gene cluster on human Ch2 (1-3). Each IL-1 family member contains an IL-1 fold. This fold is generated by 12 packed beta -sheets that interact to form a beta -trefoil structure. Little amino acid (aa) homology is required to achieve this structure, and this explains the low aa identity among IL-1 family members. IL-1ra is a pure cytokine receptor antagonist that has no signal transduction-initiating activity (4). It is an acute phase protein that exists to dampen inflammation. IL-1( beta ) is initially produced by monocytes in response to a variety of stimuli. Circulating IL-1 then binds to widely expressed IL-1 type I receptors (IL-1 RI) and initiates a number of pro-inflammatory events. On endothelial cells (EC), IL-1 induces PGE2 and IL-6 release, generating fever, thrombocytosis, and hepatic acute phase protein production. In synovial joints, IL-1 induces chondrocyte NO production, an event that leads to reduced collagen synthesis and chondrocyte apoptosis. Finally, IL-1 increases neutrophil counts, both in blood and tissue, and thus is able to promote a pro-inflammatory environment in multiple locations (5-8). IL-1ra blocks IL-1 action through competitive inhibition. More correctly, although IL-1ra fills the IL-1 binding site in IL-1 RI, it is also unable to orchestrate the creation of a signal-transducing IL-1 RI:IL-1 R Accessory protein (IL-1 R AcP) heterodimer complex. Effective IL-1ra concentrations are generally 100-fold greater than local IL-1 concentrations. This is because the IL-1ra half-life is but 6 minutes, and very few IL-1 type I receptors need to be engaged by IL-1 to elicit a cellular response (5, 7, 9). 

Human IL-1ra is synthesized as a 177 aa precursor that contains a 25 aa signal sequence and a 152 aa mature region (10, 11). Although it contains an IL-1 cytokine fold, it apparently lacks two structural motifs that allow for activation of the IL-1 receptor heterodimer. First, and following binding to IL-1 RI, the presence of Ile 51-His 54 and Lys 145 of the mature molecule preclude recruitment of IL-1 R AcP. Second, there is no identifiable C-terminal lectin segment that is hypothesized to help recruit an accessory signaling component (1, 12, 13). Mature human IL-1ra is 77% and 82% aa identical to mouse and canine IL-1ra, respectively, and human IL-1ra inhibits IL-1 activity on mouse cells (10). A number of cell types express IL-1ra, including monocytes (11), Sertoli cells (14), hepatocytes (15), adipocytes (16), synovial fibroblasts (17), mast cells (18), pancreatic beta -cells (19), and intestinal epithelial cells (20). There are at least three intracellular IL-1ra isoforms (icIL-1ra1, 2, and 3). All show N-terminal variation, and all contain amino acids 35-177 of the secreted precursor (21-23). Intracellular IL-1ra1 is of particular interest, because it is reported to be "secreted" by endothelial cells and binds to the IL-1 RI in an antagonist fashion (23-25). Intracellular IL-1ra1 is 159 aa in length and shows a 3 aa substitution for the first 21 aa's of the signal sequence of IL-1ra (21).

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.