Mouse IL-1 Beta 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.
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.
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 1 Beta (IL-1β) is a name that designates two proteins, IL-1α and IL-1β, which are the products of distinct genes, but which recognize the same cell surface receptors. With the exception of skin keratinocytes, some epithelial cells, and certain cells of the central nervous system, IL-1 is not produced by the cells of healthy individuals. However, in response to stimuli such as those produced by inflammatory agents, infections, or microbial endotoxins, a dramatic increase in the production of IL-1 by macrophages and various other cell types is seen(1-3).
IL-1α and IL-1β are structurally related polypeptides that show approximately 25% homology at the amino acid level (2). Both are synthesized as 31 kDa precursors that are subsequently cleaved into proteins with molecular weights of approximately 17.5 kDa (4, 5).   Intracellular IL-1β consists exclusively of the 31 kDa precursor form (6). Extracellular IL-1βconsists of a mixture of both unprocessed and mature IL-1β. These results indicate that processing takes place subsequently to secretion and is not tightly coupled to secretion (7). The specific protease apparently responsible for the processing of IL-1β, designated interleukin-1β-converting enzyme (ICE), has been described (7).

IL-1 possesses a wide variety of biological activities. IL-1 also plays an important role in immune functions, having effects on macrophages/ onocytes, Tlymphocytes, B lymphocytes, NK cells, and LAK cells. It acts on macrophages/ monocytes, inducing its own synthesis as well as the production of TNF and IL-6 (8,9). It activates T cells, resulting in IL-2 production and expression of IL-2 receptors (10). IL-1 also induces the production of GM-CSF and IL-4 from activated T cells (11). It induces B cell proliferation and maturation and increased immunoglobulin synthesis (12, 13).