IL-6 is a proinflammatory cytokine secreted by a variety of normal and transformed cells including T cells, B cells, monocytes/macrophages, fibroblasts, hepatocytes, keratinocytes, astrocytes, vascular endothelial cells, and various tumor cells. It plays important roles in host defense, acute phase reactions, immune responses, and hematopoiesis.
This DuoSet ELISA Development Kit is designed to measure the cytokine IL-6 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-6 (capture antibody). Upon a 1 hour blocking step, the standards (recombinant mouse IL-6) and samples are added to the wells and free IL-6 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-6 (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; we use Dako TMB Blue chromogenic substrate) 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 (i.e. when your blank wells are starting to turn blue). Color development occurs in proportion to the amount of IL-6 bound by the capture antibody. Once the development is done, the stop solution of 2N sulfuric acid (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-6 in the samples. This step can be performed by using computer software capable of generating a four parameter logistic (4PL) curve-fit. It is important to keep the level of IL-6 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-6 levels in supernatants of primary mouse macrophages, macrophage cell lines and primary microglial cells upon stimulation with pathogen-associated molecular patterns (PAMPs). 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-6 is a strongly produced cytokine in our system and we usually use a 1/4 dilution of the samples.
We have found this kit very helpful in the detection of secreted IL-6 level in the supernatants of mouse macrophages.
Instructor
Department of Medicine
University of Massachusetts