Sandwich-type enzyme immunoassay for big endothelin-I in plasma: concentrations in healthy human subjects unaffected by sex or posture.
A sandwich-type enzyme immunoassay has been developed for measuring human big endothelin-1 (big ET-1) in human plasma and supernatant fluids from human cell cultures. Big ET-1 is the precursor of endothelin 1 (ET-1), the most potent vasoconstrictor known. A rabbit antibody raised against the big ET-1 COOH-terminus fragment was used as an immobilized antibody (anti-P16). The Fab' fragment of a monoclonal antibody (1B3) raised against the ET-1 loop fragment was used as the enzyme-labeled antibody, after being coupled to acetylcholinesterase. The lowest detectable value in the assay was 1.2 pg/mL (0.12 pg/well). The assay was highly specific for big ET-1, demonstrating no cross-reactivity with ET-1, <0.4% cross-reactivity with big endothelin-2 (big ET-2), and <0.1% with big endothelin-3 (big ET-3). We used this assay to evaluate the effect of two different postural positions (supine and standing) on plasma big ET-1 concentrations in 11 male and 11 female healthy subjects. Data analysis revealed that neither sex nor body position influenced plasma big ET-1 concentrations. This assay should thus permit the detection of possible variations in plasma concentrations of big ET-1 in certain pathologies and, in association with ET-1 assay, make possible in vitro study of endothelin-converting enzyme activity in cell models. Such studies could clarify the physiological and clinical roles of this family of peptides.
Aubin
P
P
Laboratoire de Biologie Hormonale, Hôpital Saint-Louis, Paris, France.
Le Brun
G
G
Moldovan
F
F
Villette
J M
JM
Créminon
C
C
Dumas
J
J
Homyrda
L
L
Soliman
H
H
Azizi
M
M
Fiet
J
J
D000293:Adolescent; D000328:Adult; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D019332:Endothelin-1; D016232:Endothelins / Q000097:blood* / Q000737:chemistry; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques / Q000706:statistics & numerical data; D007140:Immunoglobulin Fab Fragments; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011187:Posture; D011498:Protein Precursors / Q000097:blood* / Q000737:chemistry; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012727:Sex Characteristics
D000911:Antibodies, Monoclonal; D019332:Endothelin-1; D016232:Endothelins; D007140:Immunoglobulin Fab Fragments; D010446:Peptide Fragments; D011498:Protein Precursors