Phospholipase A2 in human ascitic fluid. Purification, characterization and immunochemical detection

• Published in: Biochemical journal Vol. 278; no. 1; pp. 263 - 267
• Main Authors: KORTESUO, P. T, NEVALAINEN, T. J
• Format: Journal Article
• Language: English
• Published: Colchester Portland Press 15.08.1991
• Subjects: Analytical, structural and metabolic biochemistry; Antibodies - immunology; Antibody Specificity; Ascitic Fluid - enzymology; Biological and medical sciences; Enzymes and enzyme inhibitors; Fluoroimmunoassay; Fundamental and applied biological sciences. Psychology; Humans; Hydrolases; Immunoblotting; Leukocytes - enzymology; Molecular Weight; Phospholipases A - analysis; Phospholipases A - blood; Phospholipases A - immunology; Phospholipases A2; Human; Purification; Enzymatic activity; Ascitic fluid; Enzyme; Phospholipase A; Molecular weight
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2780263

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 Mr of approx. 10,000 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.