PHOSPHOLIPASE-A2 IN HUMAN ASCITIC FLUID - PURIFICATION, CHARACTERIZATION AND IMMUNOCHEMICAL DETECTION

A phospholipase A2 (PLA2, EC 3.1.1.4) was purified from human cell-free ascitic fluid (a-PLA2) by ion-exchange chromatography and h.p.l.c. on a reverse-phase column to apparent homogeneity. The enzyme had an M(r) of approx. 10000 as determined by SDS/PAGE. Polyclonal antibodies raised in a rabbit were specific to a-PLA2, as judged by immunoblotting. A time-resolved fluoroimmunoassay (TR-FIA) for measuring the concentration of a-PLA2 in various body fluids was developed. The detection limit of the assay was about 6 ng/ml. The antiserum did not cross-react with pancreatic secretory phospholipase A2 as measured by TR-FIA. The enzyme content was studied in various samples, including normal human serum, buffy-coat leucocytes, synovial fluid, and pancreas and spleen homogenates.