Research Scientist
Department of Neurology
University of Texas Southwestern Medical Center Dallas
IL-10 is a cytokine of the IL-10 family (IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26) which can be secreted by a large number of different cell types, including several CD4 T cell subsets and B cells. It has pleiotropic effects in immune regulation and inflammation. Mouse IL-10 is species specific and does not cross-react with human.
The eBioscience mouse IL-10 Ready-SET-Go-ELISA kit comes in different formats ranging from 2 to 20 plates and with or without the respective ELISA plates. It contains most of the required chemicals and reagents for the assay: IL-10 capture antibody, biotinylated IL-10 detection antibody, the IL-10 standard, the detection complex (horseradish peroxidase (HRP)-conjugated avidin), assay buffer, powder for the coating buffer, and tetramethylbenzidine (TMB) substrate solution. The IL-10 standard samples have to be stored in a -80°C freezer. They are single use vials which should be discarded after one thawing. The rest of the kit needs to be kept in at 4°C. The wash buffer (PBS/0.05% Tween) and stop solution (a 2N acid solution) have to be provided by the user.
96-well immunoassay plates are coated with the capture antibody in the recommended 1:1000 dilution in coating buffer (PBS) and covered with a plastic sealing film. After overnight incubation at 4°C, the plates are washed to remove unbound antibodies followed by a blocking step with the 1:5 diluted assay buffer for at least 30 minutes. During this step, all open unbound spaces on the assay plate will be covered with protein from the assay buffer. The plate is then washed again with PBS/0.05% Tween, and serially diluted standards (4000 pg/mL to 30 pg/mL) and samples are added into the respective wells for an overnight incubation at 4°C. The plate is washed again and the biotinylated detection antibody (1:1000 diluted in 1x assay buffer) is added, which binds now to the captured IL-10 protein. After 1 h incubation at room temperature, the plates are washed and avidin conjugated horseradish peroxidase is added which binds to the biotinylated antigen-antibody complex. The plates are again thoroughly washed to remove any unbound detection enzyme.
The washing during this whole assay is critical. For best results (good signal, low background), the procedure should contain 5 washes between each step with an additional soaking step included. Finally TMB substrate is added, which will be oxidized into a deep blue colored product by the HRP. After no more than 15 minutes incubation in the dark, stop solution is added which results in the forming of a yellow colored end product. The plates need to be measured at 450 nm using an ELISA plate reader.
In conclusion, this kit is a great product to measure IL-10 levels in biological fluids. I have used it with good results cell culture supernatants as well as for plasma samples.