A sequence-dependent monoclonal antibody specific for single-chain urokinase

To mimic the sequence spanning the primary site (the Lys158-Ile159 bond) cleaved by plasmin in its conversion of single-chain urokinase plasminogen activator (scuPA) to urokinase, we synthesized the peptide Cys(Acm)-Leu-Arg-Pro-Arg-Phe-Lys-Ile-Ile-Gly-Gly-Glu-Phe-Cys [Cys(Acm)scuPA(153-164)Cys]. Immunization of A/J mice with the Cys(Acm)scuPA(153-164)Cys peptide linked to hemocyanin, followed by somatic cell fusion with a myeloma cell line (SP2/0), yielded a monoclonal antibody (SCOOP1) that bound to single-chain urokinase but not to urokinase or plasmin-treated single-chain urokinase. SCOOP1 could discriminate between single-chain urokinase and urokinase by greater than three orders of magnitude. In a radioimmunoassay, Cys(Acm)scuPA(153-164)Cys completely inhibited SCOOP1 binding to single-chain urokinase, whereas an equimolar mixture of two heptapeptides comprising the amino terminal [Cys-scuPA(153-158)] and carboxy terminal [scuPA(159-164)Cys)] halves of the cleavage site peptide did not. Thus the epitope recognized by SCOOP1 includes the Lys158-Ile159 peptide bond.