Ngo T H, Verheyen S, Knockaert I, Declerck P J
Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium.
Thromb Haemost. 1998 Apr;79(4):808-12.
Two enzyme-linked immunosorbent assays (ELISAs) for the quantitation of rat plasminogen activator inhibitor-1 (PAI-1) antigen and activity, respectively, in biological fluids were developed using monoclonal antibodies raised against recombinant rat PAI-1. These assays had a lower limit of sensitivity in plasma of 0.3 and 0.15 ng/ml, respectively. The intra-assay, inter-assay and inter-dilution coefficients of variation were 9, 14 and 9%, respectively, for the antigen assay and 8, 17 and 13%, respectively for the activity assay. Assay recoveries of recombinant rat PAI-1 (5 to 20 ng/ml) added to plasma were 73 to 88% and 89 to 93% for the antigen and the activity assay, respectively. The level of PAI-1 antigen in rat plasma was 1.8 +/- 0.9 ng/ml (mean +/- SD, n = 18), with a corresponding value of 1.0 +/- 0.5 ng/ml for PAI-1 activity. In lysed platelet-rich rat plasma PAI-1 antigen was 6.0 +/- 1.0 ng/ml (n = 8) and PAI-1 activity was 2.3 +/- 0.4 ng/ml (n = 8). Endotoxin injection (0.5 mg/kg) induced a time-dependent increase of both PAI-1 antigen and PAI-1 activity levels in rat plasma. eventually resulting in a 100- to 200-fold increase (p < 0.0001 vs. baseline). A linear correlation was found between PAI-1 antigen and activity levels in normal plasma (r = 0.63, n = 18, p < 0.01) and in plasma from endotoxin-treated rats (r = 0.90, n = 35, p < 0.001). Application of these assays for the analysis of gel filtration experiments of plasma from endotoxin-treated rats demonstrated that PAI-1 antigen eluted as two peaks (with corresponding Mr of approximately 430 kDa and 61 kDa) whereas PAI-1 activity eluted as a single peak corresponding with the high molecular weight antigen form. Thus, these unique assays allowing the specific determination of rat PAI-1 antigen and rat PAI-1 activity may constitute important tools for further investigations on the pathophysiological role of PAI-1 in a variety of experimental rat models.
利用针对重组大鼠纤溶酶原激活物抑制剂 -1（PAI -1）产生的单克隆抗体，开发了两种分别用于定量生物体液中大鼠PAI -1抗原和活性的酶联免疫吸附测定（ELISA）方法。这些测定方法在血浆中的灵敏度下限分别为0.3 ng/ml和0.15 ng/ml。抗原测定的批内、批间和稀释间变异系数分别为9%、14%和9%，活性测定的相应变异系数分别为8%、17%和13%。添加到血浆中的重组大鼠PAI -1（5至20 ng/ml）的测定回收率，抗原测定为73%至88%，活性测定为89%至93%。大鼠血浆中PAI -1抗原水平为1.8±0.9 ng/ml（平均值±标准差，n = 18），PAI -1活性的相应值为1.0±0.5 ng/ml。在富含血小板的大鼠血浆裂解物中，PAI -1抗原为6.0±1.0 ng/ml（n = 8），PAI -1活性为2.3±0.4 ng/ml（n = 8）。注射内毒素（0.5 mg/kg）导致大鼠血浆中PAI -1抗原和PAI -1活性水平随时间增加，最终增加100至200倍（与基线相比，p < 0.0001）。在正常血浆（r = 0.63，n = 18，p < 0.01）和内毒素处理大鼠的血浆（r = 0.90，n = 35，p < 0.001）中，发现PAI -1抗原和活性水平之间存在线性相关性。应用这些测定方法分析内毒素处理大鼠血浆的凝胶过滤实验表明，PAI -1抗原以两个峰洗脱（相应的Mr约为430 kDa和61 kDa），而PAI -1活性以与高分子量抗原形式相对应的单峰洗脱。因此，这些能够特异性测定大鼠PAI -1抗原和大鼠PAI -1活性的独特测定方法，可能成为进一步研究PAI -1在各种实验大鼠模型中的病理生理作用的重要工具。
