MONOCLONAL ANTIBODY 13G11 RECOGNIZES PREKALLIKREIN, KALLIKREIN AND COMPLEXES OF KALLIKREIN WITH,C1-INHIBITOR AND. α2-MACR0GL0BULIN

Abstract

Abnormal prekallikrein (PK) levels in plasma can be due to decreased biosynthesis or increased activation either by surface-activated factor XIIa (e.g., in septicemia) or by other proteases (e.g., in pancreatites). To study the products of activation of PK in plasma, intact normal plasma and plasma exposed to either activating surfaces or factor XII fragment, was immunoblotted from SDS-gels. MAb 13G11 which recognizes purified PK, kallikrein (KAL) and the complexes of KAL with Cl-inhibitor (Cl-Inh) and α2macroglobulin (α2M) formed from purified proteins detected a doubLet (88- and 85-kDa) which comigrated with PK and KAL but was not visible in PK-deficient plasma. Transfer of either PK in normal plasma (25-125 ng) or KAL (50-300 ng) added to PK-deficient plasma was proportional to the amount of protein applied to the SDS-gels. Activation of plasma decreased the intensity of the PK bands with the formation of new bands with molecular weights similar to those of KAL-Cl-Inh and KAL-α2M. Identity was confirmed by MAb 4C3 (reacts with KAL-Cl-In, not with KAL) and a polyclonal antibody to α2M. Increase of incubation temperature from 24 to 37 increased KAL-Cl-Inh and decreased KAL-α2M. Addition of an excess of a2M before surface activation caused an increase of KAL-α2M complex and a decrease of KAL-Cl-Inh. Addition of an excess of Cl-Inh increased KAL-Cl-Inh and decreased KAL-α2M. In addition, activation of Cl-Inh-deficient plasma showed lower KAL-Cl-Inh and higher KAL-a2M than those when normal plasma was activated. When the deficient plasma was treated with CH3NH2 to inactivate α2M, an increase at KAL position was observed since no inhibitors were active. These studies indicate that 13G11 will be useful to detect changes in the distribution of PK, KAL, KAL-Cl-Inh and KAL-α2M associated with abnormal activation of PK and/or abnormal availability of inhibitors in disease.