LOCALIZATION OF EPITOPES RECOGNIZED BY MONOCLONAL-ANTIBODIES ON TISSUE-TYPE AND UROKINASE-TYPE PLASMINOGEN ACTIVATORS USING RECOMBINANT HYBRID ENZYMES

Hybrids of tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activator proteins expressed in CHO cells were used to locate epitopes recognized by a set of monoclonal antibodies (mAbs). Of the 19 anti-t-PA mAbs, which include 9 hitherto undescribed mAbs, one mAb did not bind to hybrids lacking the EGF-like domain. Five mAbs each recognized a distinct epitope on kringle-1. Nine other mAbs bound to at least 8 different sites on kringle-2 and 4 mAbs to at least 3 sites on the protease domain. The 4 anti-u-PA antibodies respectively bound to the growth factor domain, the kringle domain, the residual fragment of the A-chain bound to the B-chain in low-molecular weight urokinase (LMW-UK) and the protease domain. Two different mAbs which bind to partially overlapping epitopes on the t-PA kringle-1 domain both no longer reacted if Ser 119, part of an N-glycosylation signal, was mutated to Asn. However, loss of epitope recognition was not due to the absence of glycosylation. Only one mAb bound unfolded S-carboxymethylated t-PA, although several reacted efficiently with reduced sodium dodecylsulphate treated t-PA on western blots. An ELISA has been developed which detects subnanogram amounts of t-PA and kringle-2 domain-containing hybrid enzymes in water and plasma samples from animals and man and which is not inhibited by plasminogen activator inhibitor 1 (PAI-1) or protease nexin.