Mouse IL-10 ELISA Assay

Additional Information

Assay Procedure

1. Prepare all reagents and working standards as directed in the previous sections.
2. Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2-8°C sealed tightly.
3. Add 100 µL of Standard, control, or sample, per well. Cover with the adhesive strip provided. Incubate for 1.5 hours at 37°C.
4. Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (350 µL) using a squirt bottle, manifold dispenser or auto-washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
5. Add 100 µL of the working solution of Biotin-Conjugate to each well. Cover with a new adhesive strip and incubate 1 hour at 37°C.
6. Repeat the aspiration/wash as in step 3.
7. Add 100 µL of the working solution of Streptavidin-HRP to each well. Cover with a new adhesive strip and incubate for 30 minutes at 37°C Avoid placing the plate in direct light.
8. Repeat the aspiration/wash as in step 3.
9. Add 100 µL of Substrate Solution to each well. Incubate for 10-20 minutes at 37°C. Avoid placing the plate in direct light.
10. Add 100 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
11. Determine the optical density of each well immediately, using a microplate reader set to 450 nm.(optionally 630nm as the reference wave length;610-650nm is acceptable

Assay Background

Interleukin 10 (IL-10), initially designated cytokine synthesis inhibitor, is a pleiotropic cytokine that inhibits the production of a number of cytokines (including IL-1, GM-CSF, TNF, IL-6, IL-8, IL-12 and IFN-γ) by activated Th1 cells, NK cells, and monocytes/macrophages.

Mouse IL-10 is produced by a wide variety of cells, including activated Th2 cells, fetal thymocytes, monocytes/macrophages, keratinocytes, B cells, and glial cells (1-3). Mouse IL-10 cDNA encodes a 178 amino acid residue precursor protein with a hydrophobic signal peptide that is cleaved to generate the 160 amino acid residue mature protein (4). At the amino acid sequence level, there is approximately 73% identity between mouse and rat or mouse and human IL-10. Although human IL-10 is active on mouse cells, mouse IL-10 does not show species cross-reactivity on human cells (1). Herpes viruses, including EBV and equine herpes virus type 2, have been shown to encode viral homologues of IL-10 that exhibit some of the activities of IL-10 on both mouse and human cells (5,6).

IL-10 has been shown to inhibit macrophage cytotoxic activity and to stimulate the proliferation and differentiation of B cells, mast cells, and thymic T cells. IL-10 is a potent modulator of monocyte/macrophage function. IL-10 also enhances the release of soluble TNF receptors and inhibits the expression of surface ICAM-1 and B7 (7, 8). Finally, IL-10 has been reported to suppress the synthesis of superoxide anion (9) plus reactive oxygen intermediates (ROI), and either inhibit or facilitate nitric oxide synthesis, depending on the time of exposure to activated macrophages (10). IL-10 has marked effects on B cells. IL-10 also has documented activity on endothelial cells, where it mimics IL-4 (11), and on thymocytes (12) and mast cells, where it acts as a growth costimulator (13, 14).