Assay Principle 

The DetectX^® TNF-alpha Mouse ELISA Kit quantitatively measures murine TNF-alpha in serum, plasma, and tissue culture media. The kit is a sandwich ELISA with total incubation time under 4 hours. A recombinant mouse TNF-alpha standard is provided to generate a standard curve for the assay. Read all samples off the standard curve.

Protocol Summary

• Pipette standards or diluted samples into the provided clear microtiter plate coated with a polyclonal antibody to capture TNF-alpha.
• Incubate for 2 hours, followed by washing.
• Add a biotinylated TNF-alpha antibody and incubate for an additional 60 minutes.
• Perform a second wash and add peroxidase-conjugated streptavidin, followed by a 30-minute incubation.
• After a final wash, add substrate to react with the bound peroxidase-conjugated streptavidin.
• Stop the reaction and measure the color intensity at 450 nm wavelength using a microtiter plate reader. Calculate the TNF-alpha concentration in the sample using the standard curve and appropriate software.

Background

TNF-alpha (Tumor Necrosis Factor-alpha) is a key pro-inflammatory cytokine produced predominantly by macrophages and monocytes during acute inflammation. It plays a crucial role in several cellular signaling events and influences the body’s defense mechanisms against infections and cancers. However, under certain circumstances, TNF-alpha also triggers necrosis or apoptosis of host cells. Its pleiotropic effects often lead to opposing outcomes during the development of immune-mediated diseases such as cancer, Alzheimer’s, inflammatory bowel disease, Crohn’s Disease, ulcerative colitis, rheumatoid arthritis, psoriatic arthritis, psoriasis, and noninfectious uveitis. Despite this, a number of TNF-alpha therapies have been developed, and some have been approved by the FDA.

Murine models are particularly useful in biomedical research to study the role of TNF-alpha in disease processes and to test new therapeutic strategies. The ability to accurately measure TNF-alpha in mouse samples is essential for understanding its role in mouse physiology and pathology, and for the translation of these findings to human health and disease. The DetectX® TNF-alpha Mouse ELISA Kit provides a reliable and sensitive method for measuring TNF-alpha levels in these models, making it invaluable for research in immunology, inflammation, and therapeutic drug monitoring.