Rat TNF-alpha Quantikine ELISA Kit Summary

Product Summary

The Quantikine Rat TNF-alpha Immunoassay is a 4.5 hour solid phase ELISA designed to measure rat TNF-alpha levels in cell culture supernates, serum, and plasma. It contains E. coli-expressed recombinant rat TNF-alpha and antibodies raised against the recombinant factor. This immunoassay has been shown to quantitate the recombinant rat TNF-alpha accurately. Results obtained using natural rat TNF-alpha showed dose response curves that were parallel to the standard curves obtained using the recombinant kit standards. These results indicate that this kit can be used to determine relative mass values for natural rat TNF-alpha.

Preparation and Storage

Background: TNF-alpha

Tumor necrosis factor alpha (TNF-alpha ), also known as cachectin and TNFSF1A, is the prototypic ligand of the TNF superfamily (1). It is a pleiotropic molecule that plays a central role in inflammation, immune system development, apoptosis, and lipid metabolism (2-5). TNF-alpha is also involved in a number of pathological conditions including asthma, Crohn's disease, rheumatoid arthritis, neuropathic pain, obesity, type 2 diabetes, septic shock, autoimmunity, and cancer (5-11).  

Rat TNF-alpha is synthesized as a 26 kDa type II transmembrane protein that consists of a 35 amino acid (aa) cytoplasmic domain, a 21 aa transmembrane segment, and a 156 aa extracellular domain (ECD) (12). Within the ECD, rat TNF-alpha shares 95% aa sequence identity with mouse, and 73% - 79% aa identity with bovine, canine, cotton rat, equine, feline, human, rhesus macaque, and porcine  TNF-alpha. It is produced by a wide variety of immune, epithelial, endothelial, and tumor cells. TNF-alpha is assembled intracellularly to form a noncovalently linked homotrimer which is expressed on the cell surface (13). Cell surface TNF-alpha can both induce the lysis of tumor cells and virus infected cells, and generate its own downstream cell signaling following ligation by soluble TNF RI (14, 15). Shedding of membrane bound TNF-alpha by TACE/ADAM17 releases the bioactive cytokine, a 55 kDa soluble trimer of the TNF-alpha extracellular domain (16-18).  

TNF-alpha binds the ubiquitous 55 - 60 kDa TNF RI (19, 20) and the hematopoietic cell-restricted 78-80 kDa TNF RII (21, 22), both of which are also expressed as homotrimers (1, 23). Both type I and type II receptors bind TNF-alpha with comparable affinity and can promote NF kappa B activation (24-27). Only TNF RI, however, contains a cytoplasmic death domain which triggers the activation of apoptosis (3, 28). Soluble forms of both types of receptors are released into human serum and urine and can neutralize the biological activity of TNF-alpha (29-31).

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1.     Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2.     Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3.     Add 50 μL of Assay Diluent to each well.

4.     Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5.     Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6.     Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7.     Aspirate and wash 5 times.

8.     Add 100 μL Substrate Solution to each well.

9.     Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.