Novel and sensitive noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for α-human atrial natriuretic peptide in plasma

✍ Scribed by Seiichi Hashida; Naoko Yamamoto; Eiji Ishikawa

• Publisher: John Wiley and Sons
• Year: 1991
• Tongue: English
• Weight: 585 KB
• Volume: 5
• Category: Article
• ISSN: 0887-8013
• DOI: 10.1002/jcla.1860050506

✦ Synopsis

A novel and sensitive noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for a-human atrial natriuretic peptide (a-hANP) in plasma, which uses only one monoclonal IgG for the ring structure of a-hANP, is described. Plasma was filtered through polysaccharide membrane to separate peptides from proteins. The plasma filtrate was incubated with N-hydroxysuccinimidobiotin to biotinylate a-hANP and subsequently with a polystyrene ball coated with monoclonal IgG for the ring structure of a-hANP to trap biotinylated a-hANP The polystyrene ball was washed to eliminate unreacted N-hydroxysuccinimidobiotin and other biotinylated substances, and biotinylated a-hANP was eluted from the polystyrene ball with HCI. The eluate was neutralized and incubated with horseradish peroxidase-la-beled antibody IgG for the ring structure of a-hANP and subsequently with two streptavidin-coated polystyrene balls. Peroxidase activity bound to the streptavidin-coated polystyrene balls was assayed by fluorometry. The detection limit of a-hANP was 20 amol, and the assay range of plasma a-hANP was 0.8-1,200 nglL using 100 pI of plasma filtrates corresponding to 75 pl of plasma. Plasma levels of hANP in healthy subjects were 9.8-21.5 ng/L. These values were significantly lower than those measured by a two-site enzyme immunoassay probably due to the presence of a-hANPs lacking some N-terminal amino acids, which were as reactive as a-hANP [l-281 in the two-site enzyme immunoassay but less reactive in the heterotwo-site enzyme immunoassay.