Measurement of human activated Factor X-antithrombin complex by an enzyme-linked differential-antibody immunosorbent assay

✍ Scribed by Jolyon Jesty; Sidonie A. Morrison; Peter C. Harpel

• Publisher: Elsevier Science
• Year: 1984
• Tongue: English
• Weight: 848 KB
• Volume: 139
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(84)90401-9

✦ Synopsis

An enzyme-linked immunoabsorbent assay (ELBA) has been developed for the measurement of the complex of human antithrombin and Factor X.. Rabbit anti-human Factor X antibodies are adsorbed to ELISA plates, and samples containing X,-antithrombin complex are added. This is followed by the addition of F(ab'h fragments of rabbit antibodies against human antithrombin, previously labeled with alkaline phosphatase, and subsequent measurement of the bound labeled antibody by hydrolysis ofpnitrophenylphosphate. The minimum level ofcomplex detectable in a sample is ca. 0.1 nM. The assay has been used to follow the generation of X,antithrombin complex in kinetic situations by the addition of 1 pM Ile-Glu-Gly-Arg-chloromethylketone to the ELBA sampling buffer, and it has also been used in plasma systems, where a 20-fold reduction in the sensitivity of the assay is observed. This reduction was shown to be entirely caused by the plasma Factor X. The assay has been used to follow generation of the X,-antithrombin complex in defibrinated plasma upon activation of the clotting system with the Factor X-activating protein of Russell's Viper venom, and has been compared with the total generation of Factor X., measured by a radiopeptide assay of Factor X activation in the same mixtures.