A determination method of human plasma high and low molecular weight kininogens using enzyme immunoassay of bradykinin

Abstract

An enzyme immunoassay of bradykinin (BK) was developed using β-D-galactosi-dase as a labeling enzyme. The reaction of the labeled BK and the diluted anti-BK antiserum (antibody) with standard BK or sample BK was done for 30 min at 0°C. Separation of the antibody bound BK from free labeled BK was performed at 0°C for 30 min by addition of the cell wall of Staphylococcus aureus Cowan I. After centrifugation at 1000 × g at 4°C for 15 min, the activity of 3-D-galactosidase in the supernatant was measured by incubation with 2-nitrophenol β-D-galactopyranoside for 30 min at 30°C. A standard curve was obtained by adding 0.32 to 40 ng of standard BK. The antibody recognized arginine of the C-terminal of BK. The full conversion of high molecular weight (HMW), low molecular weight (LMW) and total kininogens into BK was performed by the method reported previously in THROMBOSIS RESEARCH (15: 127-134, 1979). The amounts of HMW, LMW and total kininogens in human plasmas (n = 20) were 11.010.3, 38.3±0.6 and 50.2 ±0.8 ng BK/mg protein. The levels of HMW and LMW kininogens were not changed significantly between male and female plasmas. The percentage of factor XH and plasma prekalli-krein needed to generate the maximal amount of BK from HMW kininogen was only 10%. The HMW kininogen level in the pleural exudate at 5 hr of rat carrageenin-induced pleurisy was nearly zero, even if the enzymes in the pleural exudate were supplemented with normal plasma.