Novel and ultrasensitive noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay) for α-human atrial natriuretic peptide

✍ Scribed by Seiichi Hashida; Dr. Eiji Ishikawa

• Publisher: John Wiley and Sons
• Year: 1992
• Tongue: English
• Weight: 675 KB
• Volume: 6
• Category: Article
• ISSN: 0887-8013
• DOI: 10.1002/jcla.1860060407

✦ Synopsis

A novel and ultrasensitive noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay) for a- human atrial natriuretic peptide (a-hANP) in plasma is described. a-hANP was biotinylated using N-hydroxysuccinimidobiotin and trapped onto an anti-a-hANP [6-281 IgG-coated polystyrene ball. After washing, biotinylated a- hANP was eluted from the polystyrene ball with HCI and was reacted with 2,4-dinitrophenylfluorescein-bovine serum albumin-disulfiderabbit anti-a-hANP [6-281 IgG conjugate. The complex formed was trapped onto (anti-2,4dinitrophenyl group) IgG-coated polystyrene balls and, after washing, reacted with avidinp-D-galactosidase conjugate. The polystyrene balls were washed, and the complex of the three components was eluted with EN-2, 4-dinitrophenyl-L-lysine and transferred to anti-fluorescein IgG-coated polystyrene balls. After washing, the complex was released from the polystyrene balls by reduction with 2-mercaptoethylamine and transferred to (anti-rabbit IgG) IgG-coated polystyrene balls. p-D-Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. The detection limit of a-hANP [l-281 was 3 fg (1 amol)/tube. Interference by plasma proteins was eliminated by separation of peptides from proteins using a molecular sieve. The assay range of plasma a-hANP [l-281 was 0.04-120 ng/L, and plasma levels of hANP in healthy subjects (1 1-56 ng/L) were measured without concentration.