Ishikawa E, Tanaka K, Hashida S
Department of Biochemistry, Medical College of Miyazaki, Japan.
Clin Biochem. 1990 Oct;23(5):445-53. doi: 10.1016/0009-9120(90)90238-p.
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.
本文描述了一种新型、灵敏的非竞争性（双位点）酶免疫测定法，用于测量阿托摩尔量的半抗原，尤其是小肽，这些半抗原不能被针对半抗原分子上相应两个不同表位的两种不同抗体同时结合；因此，它们不能通过传统的双位点免疫测定法进行测量。该测定法的原理是用适当的物质标记待测半抗原，使半抗原分子能够同时被标记物的结合物质分子和抗半抗原抗体分子结合，从而实现双位点测定。以生物素作为标记物，以含有10个氨基酸的单链肽血管紧张素I作为模型半抗原，证明了该原理的可行性。血管紧张素I经生物素化处理，去除未反应的生物素和其他生物素化物质后，使用抗血管紧张素I Fab'-辣根过氧化物酶偶联物和链霉亲和素包被的聚苯乙烯球进行测量。血管紧张素I的检测限为10阿托摩尔（13飞克）；这比使用相同抗体和125I-血管紧张素I的竞争性放射免疫测定法低100倍。该原理已成功应用于精氨酸加压素的测量，精氨酸加压素是一种含有9个氨基酸的单链肽，分子内有一个二硫键。本文还讨论了该测定方法可能的改进、优点和缺点。
