Tumor necrosis factor alpha (TNF alpha) is a pleiotropic inflammatory, pro-apoptotic cytokine. It exists as a homotrimer, consisting of three 17 kDa subunits, with a molecular mass of approximately 51 kDa. It regulates the activity of neutrophils, eosinophils, and lymphocytes and modulates the properties of the vascular endothelium. It is suspected to enhance or deter cellular survival through activation of receptor-mediated signal transduction. TNF alpha is known to be involved in the regulation of the body's circadian rhythm as well as in the remodeling or replacement of injured tissue. TNF alpha seems to serve as a mediator in various pathologies including septic shock, asthma, rheumatoid arthritis, trauma, diabetes, cancer, transplantation rejection and neuroinflammation-mediated brain death in Alzheimer’s disease, Parkinson’s disease, and stroke. Estimations of TNF alpha levels serve as a valuable tool to study the involvement of TNF alpha in various physiological or pathological conditions.
The eBioscience TNF alpha Ready-SET-Go!® ELISA kit offers an easy and sensitive method to quantify TNF alpha in biological samples. Using this kit, we have estimated TNF alpha levels in the plasma samples of rats subjected to cerebral ischemia. To perform the assay, the standard (100 ul/well) and plasma samples were added into the anti-rat TNF alpha precoated wells of the ELISA plate (included with the kit). The immobilized anti-TNF alpha antibody bound to the TNF alpha in the samples. After overnight incubation (4°C), wells were washed 5 times with wash buffer to remove unbound protein. The washed wells were then incubated with biotin conjugated anti-rat TNF alpha antibody (100 ul/well) for 1 h at room temperature. Wells were washed 5 times and incubated with avidin conjugated horseradish peroxidase (HRP) at room temperature for 30 minutes. Solution from the wells was aspirated and the wells were washed in order to remove excess of HRP enzyme. After washing, tetramethylbenzidine (TMB) solution was added (100 ul/well) into each well and the plate was incubated at room temperature. TMB is a substrate for HRP and gets converted to a blue colored precipitate; color development correlates with the quantity of HRP enzyme. Hence, the formation of blue color is directly proportional to the TNF alpha levels present in the samples. After 15 min, the substrate-enzyme reaction was terminated by adding stop solution which also dissolves the blue precipitate to give a soluble yellow color. The absorbance of the yellow color was read at 450 nm. The concentration of TNF alpha in the samples was calculated from the standard curve generated using the absorbance and concentration of the TNF alpha standards.
The assay procedure of this kit is reasonably simple. The results were found to consistent and reproducible. The small result variations can be minimized by analyzing the sample in triplicate. I would like to emphasize that washing is very critical and important; improper or insufficient washing may interfere with the intensity of signal. To ensure sufficient washing, wells were soaked in wash buffer for 2 minutes prior to aspiration. Following aspiration, the plate was blotted on absorbent paper to remove any residual buffer. Also, dirt spots on the bottom of the plate can affect the absorbance; hence, the bottom of the plates should be wiped before measuring the absorbance.
In summary, this kit offers a simple and sensitive method to measure TNF alpha protein levels in biological samples.