Mouse IL-1a ELISA Assay

Additional Information

Assay Principle

The Eagle Biosciences Mouse Interleukin 1 Alpha (IL-1α) ELISA Assay Kit employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-1α has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL-1α present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for IL-1α is added to the wells and binds to the combination of capture antibody-IL-1α in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A colored product is formed in proportion to the amount of IL-1α present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven IL-1α standard dilutions and IL-1α sample concentration determined.

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 Alpha (IL-1α) is a proinflammatory cytokine produced by a wide variety of cell types, including macrophages, osteoblasts, monocytes and hepatocytes. IL-1 Alpha is synthesized as 31 kDa precursors that are subsequently cleaved into proteins with molecular weights of approximately 17 kDa. The IL-1α precursor reportedly shows full biological activity in the EL-4 assay. Circulating levels of IL-1α are normally low and only rise after stimulation by agents such as those produced by inflammation, infection or microbial endotoxins. IL-1α possesses a wide variety of biological activities and exerts its effects by binding to specific cell surface receptors.