Interleukin-6 (IL-6), also known as B cell stimulatory factor 2 or interferon beta 2, is a pleotropic cytokine that provokes a broad range of cellular and physiological responses. It acts as both a pro-inflammatory and anti-inflammatory cytokine. It is secreated by T cells, macrophages, fibroblasts, endothelial cells, and keratinocytes in response to a variety of stimuli including trauma, ischemia, LPS (lipopolysaccharide), IL-1 and TNF alpha. IL-6 is relevant to many disease processes such as rheumatoid arthritis, systemic lupus erythematosus, diabetes, atherosclerosis, cerebral ischemia, Alzheimer's disease and cancer. Hence there is an interest in studying its involvement in the pathological signaling of associated disorders.
The eBioscience IL-6 Ready-SET-Go! ELISA kit offers an easy and sensitive method to quantify IL-6 levels in biological samples. This is a ready-to-use kit which contains all of the required chemicals and reagents: antibody-coated ELISA plates, detection antibody, standard, assay diluent, ELISA wash buffer powder, detection enzyme (avidin-HRP), tetramethylbenzidine (TMB) substrate solution and stop solution.
The microtiter plate is pre-coated with pre-titered amounts of IL-6 antibodies, providing binding sites for the IL-6 protein present in the samples. After overnight incubation with samples, wells are washed to remove the unbound protein. Biotinylated detection antibodies are then added, which selectively bind to the captured IL-6 protein. Unbound detection antibodies are washed away. The detection enzyme (avidin conjugated horseradish peroxidase) is then applied; it binds with the immobilized biotinylated immunocomplex. The wells are then thoroughly washed to remove any unbound detection enzyme. TMB, a substrate for HRP, is then added to the wells. TMB is oxidized into a deep blue colored product by horseradish peroxidase. A clear yellow colored product is formed after addition of the stop solution. This product is then measured at 450 nm using an ELISA plate reader.
I have used this kit to estimate IL-6 levels in plasma samples from mice subjected to cerebral ischemia. IL-6 estimation using this kit is easy to perform. Clear, concise and detailed instructions are provided with the kit. However, a few precautions can further enhance the kit's accuracy. For accurate and consistent results, the sample standards should be analyzed in triplicate. Special attention should be given while adding the samples or reagents to the wells. In no case should the tip of pipette touch to the bottom of wells. This may lead to loss of some of the antibody coating from the wells. Washing is another very critical step and insufficient washing can lead to false positive results. TMB is photosensitive and hence addition and incubation of TMB should be done in dark. The absorbance should be measured immediately after addition of stop solution. While measuring absorbance, the microplate wells should not contain air bubbles.
In summary, this is a complete, ready to use kit, for estimation of IL-6 concentrations in biological samples.