Rat IL-10 Quantikine ELISA Kit Summary

Product Summary

The Quantikine Rat IL-10 Immunoassay is a 4.5 hour solid phase ELISA designed to measure rat IL-10 levels in cell culture supernates, serum, and plasma. It contains E. coli-expressed recombinant rat IL-10 and antibodies raised against the recombinant factor. This immunoassay has been shown to quantitate the recombinant rat IL-10 accurately. Results obtained using natural rat IL-10 showed dose-response curves that were parallel to the standard curves obtained using the recombinant Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for natural rat IL-10.

Preparation and Storage

Background: IL-10

Interleukin-10 (IL-10), also known as cytokine synthesis inhibitory factor (CSIF), is the charter member of the IL-10 alpha -helical cytokine family that also includes IL-19, IL-20, IL-22, IL-24, and IL-26/AK155 (1-3). IL-10 is secreted by many activated hematopoietic cell types as well as hepatic stellate cells, keratinocytes, and placental cytotrophoblasts. Whereas human IL-10 is active on mouse cells, mouse IL-10 does not act on human cells (4, 5). Mature rat IL-10 shares 85% amino acid sequence identity with mouse IL-10 and 71% - 79% with bovine, canine, equine, feline, guinea pig, human, ovine, and porcine IL-10. It contains two intrachain disulfide bridges and is expressed as a 36 kDa noncovalently-associated homodimer (4, 6, 7). 

IL-10 mediates its biological activities through a heteromeric receptor complex composed of the type II cytokine receptor subunits IL-10 R alpha and IL-10 R beta. IL-10 R alpha is a 110 kDa transmembrane glycoprotein that is expressed on lymphocytes, NK cells, macrophages, monocytes, astrocytes, intestinal epithelial cells, cytotrophoblasts, and activated hepatic stellate cells (8-13), while the 75 kDa transmembrane IL-10 R beta is widely expressed (14, 15). The IL-10 dimer binds to two IL-10 R alpha chains, triggering recruitment of two IL-10 R beta chains (14, 15). IL-10 R beta does not bind IL-10 directly but is required for signal transduction. IL-10 R beta also associates with IL-20 R alpha, IL-22 R alpha 1, or IL-28 R alpha to form the receptor complexes for IL-22, IL-26, IL-28, and IL-29 (16-18). 

The involvement of IL-10 in immunoregulation includes both suppressive and stimulatory effects. It functions as an anti-inflammatory cytokine by inhibiting the expansion and activation of Th1 cells and Th17 cells (19-21), and by promoting the development of M2 macrophages (21). Its expression by immunosuppressive regulatory T cells (Treg) and regulatory B cells is important for Treg proliferation (19). Within a tumor microenvironment, however, IL-10 inhibits the expansion of Treg as well as myeloid-derived suppressor cells (22, 23). IL-10 induces the intratumoral accumulation and activation of CD8+ T cells (24, 25). IL-10 exerts protective effects including limiting tissue damage in arthritic inflammation (19) and promoting muscle regeneration after injury (21), but it also contributes to the persistence of viral infections (26). The levels of IL-10 have been found to be elevated in Sjogren's syndrome (saliva), primary CNS lymphoma (cerebrospinal fluid), and ovarian cancer (serum and ascites) (27-29). Its levels have been found to be decreased in the serum of patients with recurrent heart attacks or during preeclampsia and also in the seminal fluid of infertile men (30-32).

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.