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

Product Summary

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

Preparation and Storage

Background: O IL-1ra/IL-1F3

Mouse Interleukin-1 receptor antagonist (IL-1ra) is a 22-25 kDa glycoprotein produced by a variety of cell types that antagonizes IL-1 activity (1-3). It is a member of the IL-1 family of proteins that includes IL-1 alpha and IL-1 beta. Although there is little amino acid (aa) identity (< 30%) among the three IL-1 family members, all molecules bind to the same receptors, all show a beta -trefoil structure, all are physically placed on mouse chromosome # 2, and all are believed to have evolved from a common ancestral gene (1-4). Mouse IL-1ra is synthesized as a 178 aa precursor that contains a 26 aa signal sequence plus a 152 aa mature region. There is one intrachain disulfide bond and one potential N-linked glycosylation site (3, 5, 6). Mature mouse IL-1ra shares 90%, 77%, 80%, 80%, and 78% aa sequence identity to mature rat (4), human (7), porcine (8), canine (9), and equine (10) IL-1ra, respectively. In humans, three non-secreted IL-1ra isoforms have also been identified (11-14). These result from the use of alternate start sites or exon splicing. In mice, only one of the three human intracellular isoforms has been isolated. This mouse molecule is the ortholog of the 159 aa human intracellular isoform # 1 (6, 12). The mouse intracellular form differs from the secreted precursor by only three amino acids. Cells known to secrete IL-1ra include dermal fibroblasts (15), vascular smooth muscle cells (16), intestinal columnar epithelium (17), chondrocytes (18), macrophages (19), non-keratinized oral stratified squamous epithelium (20), mast cells (21), neutrophils and monocytes (22), Sertoli cells (23), and hepatocytes (24). 

There are two type I transmembrane glycoprotein receptors for IL-1ra: the bioactive 80 kDa type I IL-1 receptor (IL-1 RI), and the inert (decoy) 65 kDa type II IL-1 receptor (IL-1 RII). IL-1ra binding to IL-1 RI competitively blocks IL-1 ( alpha or beta ) binding to the same receptor. Unlike the IL-1/IL-1 RI complex, the IL-1ra/IL-1 RI complex cannot recruit the IL-1 receptor accessory protein that is required for signal transduction. This results in receptor ligation without cell activation (1, 25). IL-1ra also competitively blocks IL-1 binding to the decoy IL-1 RII. In this case, IL-1ra may actually potentiate IL-1 activity by cancelling the functions of both antagonists. 

All activities attributed to IL-1ra are explained by its role as a competitive inhibitor of IL-1 binding to IL-1 RI (1, 2, 26, 27). In general, the ratio of IL-1ra to IL-1 beta is close to 1 in both health and disease (26, 28). To achieve total abrogation of IL-1 activity, more than 95% of all IL-1 RI receptors apparently need to be occupied by IL-1ra (29). Thus, local concentrations of secreted IL-1ra may be the critical determinants of IL-1 activity. The function of intracellular IL-1ra is less clear. While it would seem to be a simple competitor of IL-1 activity, its role may be limited to that of downmodulating non-specific inflammation associated with cell debris and death (14, 27). It has no action intracellularly; only when released through cell death would it then become a functional IL-1 antagonist.

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.