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Immunoassays | Prototype

Erenna® Mouse TNFα Immunoassay Prototype Assay (Cat# 03-0060-AA)

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The Erenna® Mouse TNFα Immunoassay Evaluation Reagent Kit uses a quantitative fluorescent sandwich immunoassay technique to measure TNFα in plasma and serum samples. A capture antibody specific for TNFα has been pre-coated onto paramagnetic microparticles (MP). The user pipettes MP, standards, and samples into uncoated microplate wells. During incubation, the TNFα present in the sample binds to the capture antibody on the coated MP. Unbound molecules are washed away during the subsequent buffer exchange and wash steps. Fluor-labeled detection antibody is added to each well and incubated. This detection antibody recognizes and binds to TNFα that has been captured onto the MP. During the following wash step the MPs are transferred to a clean plate. Elution buffer is then added and incubated. The elution buffer dissociates the bound protein sandwich from the MP surface, releasing the labeled antibodies. These antibodies are separated during transfer to a final microplate. The plate is loaded into the Erenna System where the labeled molecules are detected and counted. The number of fluor-labeled detection antibodies counted is directly proportional to the amount of TNFα present in the sample when captured. The amount of TNFα in unknown samples is interpolated from a standard curve.

Assay Performance

TABLE 1. Analytical sensitivity of the Erenna® Mouse TNFα Immunoassay Prototype Assay1
Lower Limit of Detection 0.08 pg/mL
Lower Limit of Quantification2 0.49 pg/mL
Upper Limit of Quantification 500 pg/mL
Low-end CV% Range 1 - 8%
Low-end CV% Average 5%
Recommended Sample Volume 20 μL

1 See product insert for updated values
2 LLoQ = 20% CV and ± 20% recovery

FIGURE 2. Erenna® Mouse TNFα Immunoassay Evaluation Reagent Kit low-end standard curve signal (left) and curve fit (right). The Erenna® Mouse TNFα Immunoassay Evaluation Reagent Kit has a detection limit of 0.08 pg/mL, providing accurate quantification of mTNFα in only 20 μL of plasma. This assay enables better preclinical study design and long-term monitoring of individual mice over time.

Representative data shown for demonstration purposes only. Individual results may vary depending upon samples tested and protocol used.

Biology and Disease

Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine with cytotoxic activity that is primarily produced by macrophages. TNFα is involved in systemic inflammation and is a member of the group of cytokines that stimulate the acute phase reaction. When administered to animals or humans it causes inflammation, fever, cardiovascular eff ects, hemorrhage, coagulation, and acute phase responses similar to those seen during acute infections and shock states. By promoting the inflammatory response, TNFα is associated with many autoimmune disorders such as rheumatoid arthritis, ankylosing spondylitis, Crohn’s disease, psoriasis, and refractory asthma. TNFα also plays a role in cancer and regulates apoptotic cell death, cellular proliferation, tumorigenesis, and viral replication.

TABLE 2. Additional UniProtKB/Swiss-Prot Information*
Protein Name: Tumor necrosis factor
UniProtKB/Swiss-Prot ID: P06804
Protein-protein interaction database: P06804
Alternative Names: Cachectin, TNF-alpha, Tumor necrosis factor ligand superfamily member 2
Gene Names: Tnf
Synonym Gene Names: Tnfa, Tnfsf2
Function: Cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. It is mainly secreted by macrophages and can induce cell death of certain tumor cell lines. It is potent pyrogen causing fever by direct action or by stimulation of interleukin-1 secretion and is implicated in the induction of cachexia, Under certain conditions it can stimulate cell proliferation and induce cell differentiation.
Biological Processes: extracellular matrix organization, JNK cascade, positive regulation of chronic inflammatory response to antigenic stimulus, positive regulation of JNK cascade
Molecular Function: cytokine activity, tumor necrosis factor receptor binding

* Source Info from www.uniprot.org.

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