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ENZYME AMPLIFIED SENSITIVITY IMMUNOASSAY (EASIA)
PRINCIPLE AND PURPOSE OF TEST:
TNF-alpha, also named cachectin, is a 157 A.A. unglycosylated polypeptide cytokine mainly produced by activated macrophages (monocytes). It is a pleiotropic multifunctional cytokine. It has been shown that TNF can selectively lyse certain transformed cells, stimulate the proliferation of normal and neoplastic cells, inhibit hemopoiesis, and act as a differentiation factor for certain leukemia. Further, TNF mediates severe inflammatory reactions, stimulates collagenase and prostaglandin, stimulates fibroblast and endothelial cells, regulates T- and B-cells immune responses, stimulates several cytokines from normal cells and cell lines, and induces class I and class II MHC molecules.
The MEDGENIX TNF-alpha EASIA it is an immunoenzymetric assay for the quantitative measurement of human TNF-alpha in serum, plasma, cell culture medium or other biological fluids. It is a solid phase Enzyme Amplified Sensitivity Immunoassay (EASIA) performed on a microtiter plate. The assay is based on an oligoclonal system in which a blend of monoclonal antibodies (Mabs) directed against distinct epitopes of TNF-alpha are used.
Combining a number of distinct MAbs avoids hyperspecificity and allows highly sensitive assays with extended standard range and short incubation time. Samples or standards containing TNF-" react with capture monoclonal antibodies (MAbs 1) coated on the microtiter well and with monoclonal antibody (MAbs 2) labeled with horseradish peroxidase (HRP). After an
incubation period allowing the formation of sandwich (coated MAb1-TNF-alpha-MAb2-HRP), the microtiter plate is washed to remove unbound enzyme labeled antibodies, and bound enzyme labeled antibodies are measured through a chromogenic reaction. Chromogenic solution (TMB+H2O2) is added and
incubated. The reaction is stopped with addition of stop solution (H2SO4) and the plate is then read at the appropriate wavelength. The amount of substrate turnover is determined colorimeterically by measuring the absorbance which is proportional to TNF-alpha concentration. A standard curve
is plotted and TNF-alpha concentration in the sample is determined.
TNF-alpha is a potent immunoregulatory molecule having antitumoral, growth regulatory, immunomodulatory, proinflammatory, metabolic and pathogenic activities. Abnormal high levels of serum TNF-" have been described in septic shock, graft rejection parasitic infections, systemic infections
(e.g. HIV ), cancer, post hemofiltrations. Besides an insight into
pathogenesis, these determinations can aid in diagnosis (e.g. in graft rejection) and have prognostic value (e.g. in HIV infection).