HUMAN IL-12 ELISA TEST PROCEDURE

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Tony Rook
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HUMAN IL-12 ELISA TEST PROCEDURE

Please find the link for the following protocol:

HUMAN IL-12 ELISA TEST PROCEDURE

Link Reference:
http://aactg.s-3.com/pub/download/imm/il12.pdf

PRINCIPLE AND PURPOSE OF TEST

IL-12, a cytokine produced by macrophages and B lymphocytes, has multiple effects on T-cells and NK cells, including stimulation of cytotoxic activity, proliferation, and promotion of Th1
development and IFN-gamma and TNF production.

IL-12 is a disulfide-linked, 70 kDa (p70) heterodimeric glycoprotein composed of a 40 kDa (p40) subunit and a 35 kDa (p35) subunit. The p40 and p35 subunits are linked by a disulfide bridge which is essential for biological activity. These two units by themselves have no IL-12 activity, the p40 dimer has been shown to bind the IL-12 receptor and to be an IL-12 antagonist . This presents an analytical problem for either an immunoassay, which may cross react with monomeric or dimeric p40, or a bioassay, for which dimeric p40 can act as an antagonist. Quantitative measurement of IL-12 (p70) in the plasma /serum and culture supernatant fluids by ELISA can provide important information on this cytokine in various diseases states.

The Human IL-12 (p70) test kit is an enzyme immunoassay(EIA) , based on the sandwich principle, for the quantitative determination of Human IL-12 levels in human serum , plasma , or cell culture supernatant.

A monoclonal antibody(mouse) specific for Il-12 has been coated onto the microtiter plate provided in the kit. Patient Samples , controls , and standards are pipetted into the wells of the microtiter plate and incubated. During this incubation , the IL-12 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 IL-12 which is biotinylated , is
added together with Streptavidine-peroxidase into the wells and incubated. After incubation and, the well washed . The 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 IL-12 concentration of test
samples, which are run concurrently with the Standards, can be determined from the standard curve.The intensity of the color produced is porportional to the IL-12 concentration in the sample or standard.