Novel and Sensitive Noncompetitive Enzyme Immunoassay (Hetero-Two-Site Enzyme Immunoassay) for Arginine Vasopressin

A novel and sensitive noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for arginine vasopressin in plasma is described. Plasma (0.3 ml) was diluted 1.3-fold with an appropriate buffer and filtered by centrifugation in a micro-concentrator with polysaccharide membrane to eliminate plasma proteins. Arginine vasopressin in plasma filtrates was biotinylated and trapped onto anti-arginine vasopressin IgG-coated polystyrene balls. After washing the polystyrene balls to eliminate other biotinylated substances, the biotinylated arginine vasopressin was eluted from the polystyrene balls with HCl and was reacted with anti-arginine vasopressin Fab′-peroxidase conjugate. The complex formed was trapped onto streptavidin-coated polystyrene balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorometry. The detection limit of arginine vasopressin was 11 fg (10 amol)/tube. This was 45-fold lower than that by competitive enzyme immunoassay using the same antiserum as used in this study and 9 to 400-fold lower than those previously reported by competitive radioimmunoassays. The assay range of arginine vasopressin in plasma was 0.14–140 ng /l using 100 l of plasma filtrates corresponding to 75 u1 o f plasma. Plasma levels of arginine vasopressin i n 8 healthy subjects aged 25–41 yr with, adlibitum water in take and normal activity approximately 4 h after breakfast were 0.72 ± 0.22 (SD) ng /l (range, 0.42–1.04 ng /l).