Cleavage of Big Endothelin by a membrane-bound metalloproteinase, the Endothelin Converting Enzyme (ECE) leads to the active ET (1-21), a potent vasoconstrictor, and to the biological inactive C-terminal fragment (22-38). Serum, EDTA plasma, urine, saliva and cell culture supernatants are suitable for use in this assay. Dont change sample type during studies.This kit is a sandwich enzyme immunoassay for the direct determination of Endothelin in human samples.
In a first step, STD/sample/CTRL and detection antibody (monoclonal mouse anti human ET) are pipetted into the wells of the microtiter strips, which are pre-coated with polyclonal anti ET antibody. ET present in the STD/sample/CTRL binds to the pre-coated antibody in the well and forms a sandwich with the detection antibody. In the washing step all nonspecific unbound material is removed. In a second step, the conjugate (Streptavidin-HRPO) is pipetted into the wells and reacts with the detection antibody. After another washing step, the substrate (TMB Tetramethylbenzidine) is pipetted into the wells. The enzyme catalysed colour change of the substrate is directly proportional to the amount of ET. This colour change is detectable with a standard microtiter plate ELISA reader. A dose response curve of the absorbance (optical density, OD at 450 nm) vs. standard concentration is generated, using the values obtained from the standard. The concentration of ET in the sample is determined directly from the dose response curve.