Monoclonal Antibodies to Human 54,000 Molecular Weight Plasminogen Activator Inhibitor from Fibrosarcoma Cells - Inhibitor Neutralization and One-Step Affinity Purification

作者：Lene Nielsen, P.A. Andreasen, J Grøndahi-Hansen, J Y Huang, Peter Kristensen, Keld Danø · 发表于：Thrombosis and Haemostasis · 年份：1986 · DOI：10.1055/s-0038-1661523 · 被引用次数：117 · 研究领域：Protease and Inhibitor Mechanisms、Cell Adhesion Molecules Research、Blood Coagulation and Thrombosis Mechanisms

Mouse monoclonal antibodies were derived against a plasminogen activator inhibitor with a mol. wt. of approximately 54,000 (54 K) from the human fibrosarcoma cell line HT-1080. Screening for hybrids producing antibodies directed against the inhibitor was performed with enzyme-linked immunosorbent assay and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting. Four clones of hybridomas producing IgG1 antibodies were further characterized. The inhibitor was purified approximately 50-fold to homogeneity from conditioned cell culture fluid with a yield of approximately 85% by a one-step procedure using Sepharose-conjugated monoclonal antibody. In the 125I-fibrin plate assay one of the antibodies neutralized the effect of the inhibitor on urokinase-type plasminogen activator. Two of the antibodies bound complexes between urokinase-type plasminogen activator and inhibitor while the remaining two antibodies did not. The antibodies could be used for immunocytochemical localization of the inhibitor in HT-1080 cells. All four antibodies cross-reacted with a plasminogen activator inhibitor derived from cultured human umbilical cord endothelial cells.