Human IFN-γ ELISA Kit

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.

Target Information

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 Tlymphocytes 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.

Human IFN-γ is a 143 amino acid residue, 20 or 25 kDa glycoprotein that demonstrates little sequence homology to IFN-α or -β. Naturally occurring IFN-γ is found as either of two molecular weight species, differing in degree of glycosylation. Human IFN-γ apparently exists as a head-to-tail dimer in solution with the C-terminus of one monomer aligned with the N-terminus of the other monomer 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.

GENE ID 3458
SWISS PROT P01579
SYNONYMS IFG, IFI

Materials Supplied

Kit Components 96 Wells Quantity/Size
Aluminium pouches with a Microwell Plate coated with monoclonal antibody to human IFN-γ(8﹡12) 1 plate
Human IFN-γ Standard lyophilized, 4000 pg/ml upon reconstitution 2 vials
Concentrated Biotin-Conjugate anti-human IFN-γ monoclonal 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

Storage Store at 2 - 8°C

Performance Characteristics

REPEATABILITY The coefficient of variation of both intra-assay and inter-assay were less than 10%.
SENSITIVITY The minimum detectable dose was 7pg/mL.
ASSAY RANGE 31.25 - 2000 pg/mL
SPECIFICITY This assay recognizes both natural and recombinant human IFN-γ. The factors listed below were prepared at 10ng/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 human EGF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, MCP-1, TGF-β1, TGF-β2, TGF-β3, TGF-β5, VEGF, TNF-α, TNF-β
Recombinant mouse TNF-α, IFN-γ, IL-1α, IL-1β, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, GM-CSF, LIF
Other proteins IFN-γ