BIG ENDOTHELIN ELISA
|Method:||Sandwich ELISA, HRP/TMB, 12x8-well strips|
|Sample type:||Serum, plasma (EDTA, heparin, citrate)|
|Standard range:||0-3 pmol/ml (6 serum based standards)|
|Standard points:||0/0.1/0.2/0.4/1/3 pmol/l|
|Control:||1 serum based control|
|Sample size:||50 µl / well|
|Incubation time:||4 h / 1 h / 30 min|
|Unit conversion:||1 pg/ml = 0.2335 fmol/ml (MW: 4.283 kDa)|
LOD: 0.02 pmol/l (0 pmol/l + 3 SD)
Intra-assay (n=5) ≤ 5%, Inter-assay (n=10) ≤ 4%
The mean recovery of recombinant Big ET (spike 1 pmol/l; 2 pmol/l) is:
Serum (n=14): 98%; 105%
EDTA plasma (n=3) : 101%; 105%
Heparin plasma (n=3): 98%; 102%
Citrate plasma (n=3): 99%; 105%
The mean dilution linearity of endogenous Big ET with a low measuring human serum is:
For a 1+1 dilution = 90%
For a 1+3 dilution = 96%
For a 1+7 dilution = 98%
ET1/2/3 (1-21): <1%, ET2 (1-37): <1%, ET1/2 (1-38): <1%, porcine BigET (1-39): 21%, BigET1/2 (22-38): <1%, BigET2 (22-37): <1%, rat BigET1 (1-39): 10%, Sarafotoxin: <1%
Values from apparently healthy individuals:
Median (serum, n=41): 0.09 pmol/l
It is recommended to establish the normal range for each laboratory.
Principle of the assay:
Manual ELISAs - easily adaptable for automation!
INSTRUCTIONS FOR USE
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Big Endothelin-1 (BigET) is a peptide of 38 amino acids and is the precursor of Endothelin-1 (ET), represented by amino acids 1-21 (http://www.uniprot.org/uniprot/P05305). ET is a potent vasoconstrictor and is produced by vascular endothelial cells. Accordingly it has a wide tissue distribution (http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Hs.511899). The cleavage of BigET by Endothelin Converting Enzyme (ECE) leads to ET and to a C-terminal fragment. Both BigET and ET are strong independent predictors of survival in patients with congestive heart failure, and identify a population with a very high risk mortality. The half-life of ET (1-21) in plasma is less than one minute, whereas clearance of BigET is much slower. BigET can therefore be determined more easily.
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