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Thromb Haemost 1986; 55(02): 206-212
DOI: 10.1055/s-0038-1661523
Original Article
Schattauer GmbH Stuttgart

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

L S Nielsen
The Finsen Laboratory, Finsen Institute, and Laboratory of Tumor Biology, Institute of Pathology, University of Copenhagen, Copenhagen, Denmark
,
P A Andreasen
The Finsen Laboratory, Finsen Institute, and Laboratory of Tumor Biology, Institute of Pathology, University of Copenhagen, Copenhagen, Denmark
,
J Grøndahi-Hansen
The Finsen Laboratory, Finsen Institute, and Laboratory of Tumor Biology, Institute of Pathology, University of Copenhagen, Copenhagen, Denmark
,
J Y Huang
The Finsen Laboratory, Finsen Institute, and Laboratory of Tumor Biology, Institute of Pathology, University of Copenhagen, Copenhagen, Denmark
,
P Kristensen
The Finsen Laboratory, Finsen Institute, and Laboratory of Tumor Biology, Institute of Pathology, University of Copenhagen, Copenhagen, Denmark
,
K Danø
The Finsen Laboratory, Finsen Institute, and Laboratory of Tumor Biology, Institute of Pathology, University of Copenhagen, Copenhagen, Denmark
› Author Affiliations
Further Information

Publication History

Received 01 November 1985

Accepted 30 January 1986

Publication Date:
18 July 2018 (online)

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Summary

Mouse monoclonal antibodies were derived against a plasminogen activator inhibitor with a mol.wt. of ∼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 ∼50-fold to homogeneity from conditioned cell culture fluid with a yield of ∼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.

Key words

Plasminogen activator inhibitor - Monoclonal antibody - Fibrosarcoma cells - Inhibitor neutralization - Purification
 

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