HUMAN INTERFERON-y ELISA TEST PROCEDURE

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Tony Rook
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HUMAN INTERFERON-y ELISA TEST PROCEDURE

Please find the link for the following protocol:

HUMAN INTERFERON-y ELISA TEST PROCEDURE

Link Reference:
http://aactg.s-3.com/pub/download/imm/imifngam.pdf

PRINCIPLE AND PURPOSE OF TEST

Interferon gamma (IFN-y) is a basic , 143 amino acid glycoprotein with two potential glycosylation sites in positions 25 and 97 and dose not contain a disulfide bridge. IFN-y also
termed immune or type II IFN, is produced by several subsets of T Cells as well as by large granular lymphocytes(LGL). Like other IFNs , IFN-y acts on many cells types to induce an
antiviral state and promotes cell differentiation at the expense of cellular proliferation. Additionally , IFN-( acts as a powerful macrophage activator and induces increased expression of
cell membrane MHC II antigen, e.g., DR or Ia , on many cells types. IFN-y also acts to augment the production of other monokines such as IL-1 and TNF and thus participates in the cytokine cascade. IFN-y is a cytokine noted for its rapid production in association with immune defense responses.
It is one of the earliest trigger cytokines that appears within a few hours after infection. Quantitative measurement of IFN-y in the plasma /serum by ELISA can provide important
information on this cytokine in various diseases states.

The Human IFN-y test kit is an enzyme immunoassay(EIA) , based on the sandwich principle, for the quantitative determination of Human interferon gamma (IFN-y) levels in human serum , plasma , or cell culture supernatant.
A monoclonal antibody(mouse) specific for IFN-y has been coated onto the microtiter plate provided in the kit. Sample diluent , patient Samples , controls , and standards are pipetted into the wells of the micro titer plate and incubated. During this incubation , the IFN-y present in the specimens is bound to micotiter plate. Unbound material present in the specimens is removed by aspiration of the wells and washing them. The second monoclonal antibody to IFN-y which is biotinylated , is added to the wells and cover the plate and incubated. After incubation and washing , Streptavidine-peroxidase is added into the wells and incubated. After incubation and washing . TMB (3,3,5,5 Tetramethylbenzidine ) solution is added into all wells and incubated. The enzyme reaction is stopped by the addition of Stop solution and the absorbance at 450 nm is
measured with a spectrophotometer.

A standard curve is obtained by plotting the optical density ( absorbances ) versus the corresponding concentrations of defined standards. The human IFN-y concentration of test samples, which are run concurrently with the Standards, can be determined from the standard curve.

Detectable levels of human IFN-y are generally not found in the serum of normal healthy individuals .In the course of several diseases, some without apparent viral origin , IFNs can
present in quantities measurable in the circulation . This is the case for the disease states accompanied by immune dysfunction, which are frequently characterized by the presence of IFN-y and / or IFN-