Principle of the Assay
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IFN-γ has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IFN-γ present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for IFN-γ is added to the wells and binds to the combination of capture antibody-IFN-γ in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A coloured product is formed in proportion to the amount of IFN-γ present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven IFN-γ standard dilutions and IFN-γ sample concentration determined..
For Use with serum, plasma and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.
Interferon-gamma (IFN-γ) is a multifunctional protein first observed as an antiviral activity in cultures of Sindbis virus-infected human leukocytes stimulated by PHA. Produced by T-lymphocytes and natural killer (NK) cells, IFN-γ is now known to be both an inhibitor of viral replication and a regulator of numerous immunological functions. Human IFN-γ is reported to be active only on human and non-human primate cells. The biochemistry and biological activities of interferons have been extensively reviewed.
Mouse IFN-γ encodes a 155 amino acid (aa) residue precursor protein with a hydrophobic signal peptide that is cleaved to generate the 133 aa residue mature protein. In solution, IFN-γ has been shown to exist as a non-covalently associated homodimer with topological similarity to IL-10. Mouse IFN-γ shows approximately 40% aa sequence identity to human IFN-γ and there is no cross-reactivity across species.
A receptor for IFN-γ has been identified and its gene localized to chromosome 6 Apparently the product of a single gene, the receptor is a single chain 90 kDa glycoprotein that shows a high degree of species-specific binding of IFN-γ.
Functionally, IFN-γ produces a variety of effects. Produced by CD8+, NK, gd, and TH1 T helper cells, IFN-γ has documented antiviral, antiprotozoal and immunomodulatory effects on cell proliferation and apoptosis, as well as the stimulation and repression of a variety of genes he antiprotozoal activity of IFN-γ against Toxoplasma and Chlamydia is believed to result from indoleamine 2,3-dioxygenase activity, an enzyme induced by IFN-γ. The immunomodulatory effects of IFN-γ are extensive and diverse. In monocyte/macrophages, the activities of IFN-γ include: increasing the expression of class I and II MHC antigens; increasing the production of IL-1, platelet-activating factor, H2O2, and pterin; protection of monocytes against LAK cell-mediated lysis; downregulation of IL-8 mRNA expression that is upregulated by IL-2; and, with lipopolysaccharide, induction of NO production. Finally, IFN-γ has been shown to upregulate ICAM-1, but not E-Selectin or VCAM-1, expression on endothelial cells.
|Kit Components||96 Wells Quantity/Size|
|Aluminum pouches with a Microwell Plate coated with monoclonal antibody to mouse IFN-γ(8﹡12)||1 plate|
|Mouse IFN-γStandard lyophilized, 2000 pg/ml upon reconstitution||2 vials|
|Concentrated Biotin-Conjugate anti-mouse IFN-γmonoclonal antibody||2 vials|
|Streptavidin-HRP solution||2 vials|
|Standard /sample Diluent||1 bottle|
|Biotin-Conjugate antibody Diluent||1 bottle|
|Streptavidin-HRP Diluent||1 bottle|
|Wash Buffer Concentrate 20x (PBS with 1% Tween-20)||1 bottle|
|Substrate Solution||1 vial|
|Stop Solution||1 vial|
|Adhesive Films||4 pieces|
|Product data sheet||1 copy|
|Storage||Store at 2 - 8°C|
|REPEATABILITY||The coefficient of variation of both intra-assay and inter-assay were less than 10%.|
|SENSITIVITY||The minimum detectable dose was 4pg/mL.|
|ASSAY RANGE||7.8 - 500 pg/mL|
|SPECIFICITY||This assay recognizes both natural and recombinant mouse IFN-γ. The factors listed below were prepared at 50ng/ml in Standard /sample Diluent and assayed for cross-reactivity and no significant cross-reactivity or interference was observed.|
Factors assayed for cross-reactivity
|Recombinant mouse||G-CSF, GM-CSF, C10, TNF-α, IL-1α, IL-1β, IL-2 , IL-3, IL-5, IL-6, IL-7, IL-9, LIF, M-CSF, SCF, VEGF, MIP-2|
Data Analysis Assistance
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