HUMAN RANTES ELISA TEST PROCEDURE

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Tony Rook
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HUMAN RANTES ELISA TEST PROCEDURE

Please find the link for the following protocol:

HUMAN RANTES ELISA TEST PROCEDURE

http://aactg.s-3.com/pub/download/imm/rante.pdf

PRINCIPLE AND PURPOSE OF TEST

RANTES (regulated upon activation, normal T cell expressed and secreted) is a chemokine that belongs to the beta-chemokine family. It is a chemoattractant for CD4+/CD45RO T cells and is produced by various cell types including CD8+ and CD4+ T cells as well as monocytes and macrophages. Recently, RANTES
has been shown to supress replication of macrophage-tropic strains of HIV in CD4+ T cells.

The Human RANTES test kit is an Enzyme Linked Immunosorbent Assay(ELISA) , based on the sandwich principle, for the quantitative determination of Human RANTES levels in human serum , plasma, or cell culture supernatant. An antibody specific for RANTES 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 RANTES present in the specimens is bound to the micotiter plate. Unbound material present in the specimens is removed by aspiration of the wells and washing them. The second antibody to RANTES (which is biotinylated) is added to the plate; the
plate is then covered and incubated. After incubation and washing, Streptavidin-peroxidase is added into the wells and incubated. Once this incubation is over, the plate is again aspirated, washed. TMB (3,3=,5,5=Tetramethylbenzidine ) solution is added to all the wells (to produce a color change) and the plate 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 (absorbencies ) versus the corresponding concentrations of defined standards. The human RANTES concentration of test samples, which are run concurrently with the Standards, can be determined from the
standard curve.