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Sandwich-type enzyme immunoassay for big endothelin-1 in plasma: concentrations in healthy human subjects unaffected by sex or posture

¹ Laboratoire de Biologie Hormonale, Hôpital Saint-Louis, 75010 Paris, France.

² CEA Saclay, Service de Pharmacologie et d'Immunologie, 91191 Gif/Yvette, France.

³ Biotechnology Department, Roussel-Uclaf, 93235 Romainville, France.

⁴ Centre d'Investigation Clinique, INSERM, Hôpital Broussais, 75014 Paris, France.
^a Author for correspondence. Fax +33 1 42494280.

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.

Key Words: indexing terms: reference values • endothelins • isopeptides • vasoconstrictors • blood pressure • variation, source of

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