IL-1beta is a proinflammatory cytokine produced in a variety of cells including monocytes, tissue macrophages, keratinocytes and other epithelial cells. The release of a mature IL-1beta in the supernatant results from the processing of the intracellular IL-1beta precursor (pro- IL-1beta) by caspase-1.
This DuoSet ELISA Development Kit is designed to measure the cytokine IL-1beta in mouse cell culture supernatants. The assay takes 5-6 hours to perform after an initial overnight incubation with the capture antibody. The plate (not provided) is first coated overnight with a rat monoclonal antibody specific for mouse IL-1beta (capture antibody). Upon a 1 hour blocking step, the standards (recombinant mouse IL-1beta) and samples are added to the wells and free IL-1beta is bound by the immobilized capture antibody during a 2 hour incubation. After extensive washing of the microplate to remove any unbound substances, a biotinylated goat antibody specific for mouse IL-1beta (detection antibody) is added to the plate for another 2 hours. Upon extensive washing to remove any unbound detection antibody, a short incubation is required with a solution containing streptavidin conjugated to horseradish-peroxidase (HRP). This step requires an incubation of 20 minutes during which the plate should be kept away from direct light. Finally, after three washes, a substrate solution (not provided) is added to the wells and color (blue) development is allowed to proceed for 20 minutes maximum; it is important to check the plate in order to prevent over development of the color. Color development occurs in proportion to the amount of IL-1beta bound by the capture antibody. Once the development is done, the stop solution (not provided) is added to the well still containing the developed solution (the color turns from blue to yellow) and the optical density of each well is immediately determined using a microplate reader at 450 nm. A standard curve is created from the standards on the plate and is used to calculate the concentration of IL-1beta in the samples. This step can be performed by reducing the data using computer software capable of generating a four parameter logistic (4PL) curve-fit. It is important to keep the level of IL-1beta within the dynamic range of the assay (the standards cover the range 1000-15.6 pg/ml).
We have used this assay to measure IL-1beta level in supernatant of primary mouse macrophages and macrophage cell lines (RAW 264.7 cells) upon bacterial infection. The solutions not provided with the kit, such as the wash buffer, block buffer and the reagent diluent, are easy to prepare and require common reagents (PBS, BSA and Tween20). The substrate solution and stop solution, however, have to be acquired independently of the DuoSet Kit. The general protocol provided with the kit recommends the volume of the assay to be 100 ul/well, but we routinely use 50 ul/well without any loss in the quality of detection of IL-1beta in our sample. Used with a volume of 50 ul, this kit contains enough reagents to realize thirty 96-well plates. Il-1beta is not a strongly produced cytokine in our system and we usually use a ½ dilution of the samples.
We have found this kit very helpful in the detection of secreted IL-1beta level in the supernatant of mouse macrophages.