MacGregor I R, Booth N A
Scottish National Blood Transfusion Service, Headquarters Unit Laboratory, Edinburgh, UK.
Thromb Haemost. 1988 Feb 25;59(1):68-72.
A two-site sandwich ELISA was developed to measure PAI-1 antigen and utilised a polyclonal antiserum produced against PAI-1 purified from human endothelial cell secretory products. The assay was calibrated against a preparation of pure PAI-1 whose protein concentration had been determined by amino acid analysis and the detection limit was 30 pg PAI-1 ml-1 sample. PAI-1 was detected in primate sera but not in a wide range of non-primate sera and no cross-reactivity with alpha 2-antiplasmin or antithrombin III was observed. The ELISA was used to study cellular secretion of PAI-1 which was confirmed as a major secretory protein in human umbilical vein endothelial cells (HUVEC). PAI-1 antigen accumulated in the medium in a linear fashion with time and accounted for approximately equal to 10% of total secreted protein. Specific activity of intracellular PAI-1 was typically 20-fold greater than that of PAI-1 in 24 h conditioned medium and a t1/2 for inactivation of secreted PAI-1 of 0.53 h was calculated. Purified endotoxin stimulated the secretion of PAI-1 antigen and raised the intracellular levels in HUVEC cultures showing that the anti-fibrinolytic actions of endotoxin are effected by increasing the rate of synthesis and secretion of PAI-1.
开发了一种双位点夹心酶联免疫吸附测定法（ELISA）来检测纤溶酶原激活物抑制因子-1（PAI-1）抗原，并使用了针对从人内皮细胞分泌产物中纯化的PAI-1产生的多克隆抗血清。该测定法以一种纯PAI-1制剂进行校准，其蛋白质浓度已通过氨基酸分析确定，检测限为30 pg PAI-1 ml-1样品。在灵长类动物血清中检测到了PAI-1，但在多种非灵长类动物血清中未检测到，并且未观察到与α2-抗纤溶酶或抗凝血酶III的交叉反应。该ELISA用于研究PAI-1的细胞分泌，结果证实PAI-1是人脐静脉内皮细胞（HUVEC）中的一种主要分泌蛋白。PAI-1抗原在培养基中随时间呈线性积累，约占总分泌蛋白的10%。细胞内PAI-1的比活性通常比24小时条件培养基中的PAI-1高20倍，计算出分泌型PAI-1的失活半衰期为0.53小时。纯化的内毒素刺激了PAI-1抗原的分泌并提高了HUVEC培养物中的细胞内水平，表明内毒素的抗纤溶作用是通过增加PAI-1的合成和分泌速率来实现的。
