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A sequence-dependent monoclonal antibody specific for single-chain urokinase.
J R Charpie1, E Haber, G R Matsueda
1Cellular and Molecular Research Laboratory, Massachusetts General Hospital, Boston 02114.
Biochemical and Biophysical Research Communications
|April 29, 1988
Summary
Researchers developed a monoclonal antibody, SCOOP1, that specifically detects single-chain urokinase plasminogen activator (scuPA) by recognizing the Lys158-Ile159 peptide bond crucial for its activation.
Area of Science:
- Biochemistry
- Immunology
- Molecular Biology
Background:
- Single-chain urokinase plasminogen activator (scuPA) is a zymogen that requires cleavage by plasmin at the Lys158-Ile159 bond to become active urokinase.
- Distinguishing between scuPA and active urokinase is critical for understanding and potentially modulating fibrinolytic pathways.
Purpose of the Study:
- To generate a specific antibody that recognizes the activation site of scuPA.
- To characterize the binding properties of the generated antibody and define its epitope.
Main Methods:
- Synthesis of a peptide mimicking the scuPA cleavage site (Cys(Acm)scuPA(153-164)Cys).
- Immunization of mice with the peptide-hemocyanin conjugate and subsequent hybridoma production.
- Characterization of monoclonal antibody SCOOP1 binding to scuPA and urokinase using radioimmunoassay.
Main Results:
- Monoclonal antibody SCOOP1 was successfully generated, showing high specificity for scuPA over urokinase.
- SCOOP1 demonstrated a binding discrimination of over three orders of magnitude between scuPA and urokinase.
- Competitive inhibition assays indicated that the epitope recognized by SCOOP1 encompasses the Lys158-Ile159 peptide bond.
Conclusions:
- The monoclonal antibody SCOOP1 specifically recognizes the activation site of scuPA, including the scissile Lys158-Ile159 peptide bond.
- SCOOP1 serves as a valuable tool for differentiating between single-chain urokinase plasminogen activator and its active form, urokinase.