Measurement of Different Forms of Tissue Plasminogen Activator in Plasma

组织纤溶酶原激活剂 纤溶酶原激活剂 化学 纤溶酶原激活剂 多克隆抗体 分子生物学 结合 色谱法 抗体 生物化学 免疫学 内科学 医学 生物 数学 数学分析
作者
Wayne L. Chandler,Marcy L. Jascur,P. Henderson
出处
期刊:Clinical Chemistry [American Association for Clinical Chemistry]
卷期号:46 (1): 38-46 被引量:32
标识
DOI:10.1093/clinchem/46.1.38
摘要

Abstract Background: We evaluated assays to measure both total tissue plasminogen activator (tPA) and the three principle forms of tPA in plasma: active tPA, tPA complexed with plasminogen activator inhibitor type 1 (PAI-1), and tPA complexed with C1-inhibitor. Methods: Active tPA was measured by use of an indirect amidolytic assay and immunofunctional assays. tPA/PAI-1, tPA/C1-inhibitor, and total tPA antigen were measured by use of microtiter plates coated with anti-tPA antibodies and, respectively, anti-PAI-1, anti-C1-inhibitor, and anti-tPA antibodies conjugated to peroxidase. Results: The immunofunctional tPA assay detected 1 U/L (0.001 U/mL) tPA and recovered 108% ± 12% of active tPA added to samples containing high (mean, 60 000 IU/L) PAI-1 activities vs a detection limit of 10 U/L (0.01 U/mL) and 13% ± 25% recovery for the indirect amidolytic tPA activity assay. For measurement of tPA/PAI-1 complex, polyclonal anti-PAI-1 conjugates recovered 112% ± 20% of the expected tPA/PAI-1 vs recovery of only 38% ± 16% when monoclonal anti-PAI-1 conjugates were used. Of three methods tested, two total tPA antigen assays correlated well (r2 = 0.85) and showed recoveries near 100%, whereas the third method showed lower correlations, higher intercepts, and falsely high recovery. A single anti-tPA capture antibody that performed the best in the individual assay evaluations was used to measure the different forms of tPA in 22 samples with a range of tPA and PAI-1 values. The sum of the molar concentrations of active tPA, tPA/PAI-1, and tPA/C1-inhibitor using the optimized methods was equal to 94% ± 7% of measured total tPA. Conclusion: Optimized assays based on a single anti-tPA capture antibody can be used to accurately measure the major forms of tPA in plasma.
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