Nieman C J, Kerry R, Coleman P J, Taylor A, Jose J, Pay G F
Ciba-Geigy Pharmaceuticals, Horsham, West Sussex, UK.
Thromb Res. 1991 May 1;62(3):189-97. doi: 10.1016/0049-3848(91)90192-y.
A colourimetric enzyme-linked sandwich assay has been developed to investigate the binding of human platelets to fibrinogen. The presence of platelets bound to fibrinogen-coated plastic can easily be detected and quantitated. Platelets treated with chymotrypsin to expose the fibrinogen receptor, are fixed with paraformaldehyde, and stored frozen. The detection sandwich consists of a mouse monoclonal antibody directed against the human platelet CD9 antigen, and a rabbit anti-mouse immunoglobulin conjugated to the enzyme alkaline phosphatase. The cleavage of the phosphatase substrate p-nitrophenyl phosphate can be monitored colourimetrically. The data presented provide evidence that this method is capable of detecting platelet-fibrinogen binding in a physiologically relevant manner. The binding is inhibited by EDTA or excess fibrinogen. The fibrinogen alpha and gamma chain peptides, RGDS and LGGAKQAGDV, and the snake venom echistatin are also inhibitory with IC50 values of 135 microM, 1.8 mM and 100 nM respectively.
已开发出一种比色酶联夹心测定法来研究人血小板与纤维蛋白原的结合。结合在包被有纤维蛋白原的塑料上的血小板的存在易于检测和定量。用胰凝乳蛋白酶处理以暴露纤维蛋白原受体的血小板，用多聚甲醛固定，并冷冻保存。检测夹心由针对人血小板CD9抗原的小鼠单克隆抗体和与碱性磷酸酶酶偶联的兔抗小鼠免疫球蛋白组成。磷酸酶底物对硝基苯磷酸的裂解可以通过比色法监测。所呈现的数据提供了证据，表明该方法能够以生理相关的方式检测血小板 - 纤维蛋白原结合。这种结合受到EDTA或过量纤维蛋白原的抑制。纤维蛋白原α链和γ链肽RGDS和LGGAKQAGDV以及蛇毒echistatin也具有抑制作用，IC50值分别为135 microM、1.8 mM和100 nM。
