Novel and sensitive noncompetitive (two-site) immunoassay for haptens with emphasis on peptides

A novel and sensitive noncompetitive (two-site) Enzyme immunoassay is described to measure attomole amounts of haptens, especially small Peptides, which cannot be bound simultaneously by two different Antibodies directed to the corresponding two different epitopes on the Hapten molecules; consequently, they cannot be measured by the conventional two-site immunoassays. The principle of the assay is to label haptens to be measured with an appropriate substance, so that the Hapten molecule can be bound simultaneously by both a binding substance molecule for the label, and anti-hapten antibody molecule, which allows two-site assay. Feasibility of the principle was demonstrated by using biotin as a label and angiotensin I, a 10 amino acid single chain peptide, as a model Hapten. Angiotensin I was biotinylated and, after removal of unreacted biotin and other biotinylated substances, was measured using anti-angiotensin I Fab'-horseradish peroxidase conjugate and streptavidin-coated polystyrene balls. The detection limit of angiotensin I was 10 amol (13 fg); this was 100-fold lower than that by competitive radioimmunoassay using the same antibody and 125I-angiotensin. I. This principle was successfully applied to the measurement of arginine vasopressin, a nine amino acid single chain peptide with an intramolecular disulfide bridge. Possible modifications, advantages, and disadvantages of the assay method are discussed.