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Immunoassays | Inflammation & Cytokines

Erenna® IFN-γ Immunoassay Kit (Cat# 03-0049-xx)

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The Erenna® IFN-γ Immunoassay Kit uses a quantitative fluorescent sandwich immunoassay technique to measure IFN-γ in plasma and serum samples. A capture antibody specific for IFN-γ has been pre-coated onto paramagnetic microparticles (MP). The user pipettes MP, standards, and samples into uncoated microplate wells. During incubation, the IFN-γ 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 IFN-γ 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 IFN-γ present in the sample when captured. The amount of IFN-γ in unknown samples is interpolated from a standard curve.

Assay Performance

TABLE 1. Analytical sensitivity of the Erenna® IFN-γ Immunoassay Kit1
Lower Limit of Detection 0.04 pg/mL
Lower Limit of Quantification2 0.2 pg/mL
Upper Limit of Quantification 25 pg/mL
Low-end CV% Range 1 - 12%
Low-end CV% Average 8%
Recommended Sample Volume 100 μL
Minimum Sample Volume Required3 100 μL

1 See product insert for updated values
2 LLoQ = 20% CV and ± 20% recovery
3 based upon median [IFN-γ] in a healthy reference

FIGURE 1. [IFN-γ] in EDTA plasma from 12 healthy donors, with median and interquartile range. The Erenna® IFN-γ Immunoassay Kit reliably quantifies IFN-γ in healthy subjects, who have a median [IFN-γ] of 1.03 pg/mL that is well above the detection limit of 0.04 pg/mL.

FIGURE 2. Erenna® IFN-γ Immunoassay Kit low-end standard curve signal (left) and curve fit (right).

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

Biology and Disease

Interferon-gamma (IFN-γ) is a cytokine with antiviral and tumor-suppressing properties that is actively involved in the innate immune system. As the only member of the Type 2 IFN family, IFN-γ is expressed by Th1 cells, Tc cells, dendritic cells and natural killer cells. Once bound to its heterodimeric receptor IFN-γR, the activated glycoprotein recruits and directs macrophages to the site of infection and escalates the inflammatory response. IFN-γ has also been documented to up-regulate the expression of IL-1 and down-regulate the production of IL-8, both of which are pro-inflammatory cytokines. The potency, specificity and rapid action of IFN-γ make it an attractive drug target for immuno-regulatory diseases.

TABLE 2. Additional UniProtKB/Swiss-Prot Information*
Protein Name: Interferon gamma
UniProtKB/Swiss-Prot ID: P01579
Protein-protein interaction database: P01579
Alternative Names: Immune interferon
Gene Names: IFNG
Synonym Gene Names:
Function: Produced by lymphocytes activated by specific antigens or mitogens. IFN-gamma, in addition to having antiviral activity, has important immunoregulatory functions. It is a potent activator of macrophages, it has antiproliferative effects on transformed cells and it can potentiate the antiviral and antitumor effects of the type I interferons.
Biological Processes: negative regulation of smooth muscle cell proliferation, positive regulation of interleukin-12 production, regulation of insulin secretion, response to virus
Molecular Function: cytokine activity, interferon-gamma receptor binding

* Source Info from www.uniprot.org.

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