Novel and ultrasensitive noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay) for alpha-human atrial natriuretic peptide.
A novel and ultrasensitive noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay) for alpha-human atrial natriuretic peptide (alpha-hANP) in plasma is described. alpha-hANP was biotinylated using N-hydroxysuccinimidobiotin and trapped onto an anti-alpha-hANP [6-28] IgG-coated polystyrene ball. After washing, biotinylated alpha-hANP was eluted from the polystyrene ball with HCI and was reacted with 2,4-dinitrophenyl-fluorescein-bovine serum albumin-disulfide-rabbit anti-alpha-hANP [6-28] IgG conjugate. The complex formed was trapped onto (anti-2,4-dinitrophenyl group) IgG-coated polystyrene balls and, after washing, reacted with avidin-beta-D-galactosidase conjugate. The polystyrene balls were washed, and the complex of the three components was eluted with epsilon N-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. beta-D-Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. The detection limit of alpha-hANP [1-28] 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 alpha-hANP [1-28] was 0.04-120 ng/L, and plasma levels of hANP in healthy subjects (11-56 ng/L) were measured without concentration.
Hashida
S
S
Department of Biochemistry, Medical College of Miyazaki, Japan.
Ishikawa
E
E
D009320:Atrial Natriuretic Factor / Q000097:blood* / Q000592:standards; D001665:Binding Sites; D001710:Biotin; D006801:Humans; D007124:Immunoenzyme Techniques / Q000706:statistics & numerical data; D010446:Peptide Fragments / Q000097:blood; D012015:Reference Standards; D012680:Sensitivity and Specificity
D010446:Peptide Fragments; D001710:Biotin; D009320:Atrial Natriuretic Factor