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Time-resolved fluoroimmunoassay was developed for the detection of synovial-type phospholipase A2 (s-PLA2) in human serum. This solid-phase, sandwich assay uses a polyclonal rabbit antibody raised against synovial-type group II PLA2 produced in Escherichia coli. No cross-reactions were detected between s-PLA2 and PLA2 from human or porcine pancreas, human ascitic fluid, or bee or cobra venom. In healthy individuals, the average concentration of s-PLA2 is 3.7 micrograms/L, with a 95% reference interval from 1.3 to 10.8 micrograms/L. We investigated pancreatic PLA2, which is a group I PLA2, and synovial-type group II PLA2 in sera of patients with hematological malignancies and septic fever. The concentration of s-PLA2 was increased in patient sera and correlated significantly with the catalytic activity of PLA2 and the concentration of C-reactive protein. No correlation with the concentration of pancreatic PLA2 was found. The results suggest that the increased catalytic activity of PLA2 in sera of patients with septic fever results from synovial-type group II PLA2.

Summary:A monoclonal antibody, designated 2E1, against human pancreatic phospholipase A 2 was produced by hybridization of myeloma cells with spieen cells of immunized BALB/c mice. The hybridomas were screened for antibody production by time-resolved fluoroirnrnunoassay (TR-FIA). The antibody was found to belong to subclass I of murine IgG. The specificity of the antibody was confirmed by immunohistochemistry of pancreatic and other tissues, by immunoblotting of a crude aqueous extract of human pancreas and purified human pancreatic phospholipase A 2 and by TR-FIA. A solid-phase time-resolved fluoroimmunoassay was developed by using the monoclonal anti-phospholipase A 2 antibody äs the catching antibody and a polyclonal sheep anti-phospholipase A 2 antibody labelled with europium äs the detecting antibody. The validity of the new TR-FIA of human pancreatic phospholipase A 2 was confirmed by using it to measure the phospholipase A 2 concentrations in serum samples from healthy subjects and from patients suffering from acute pancreatitis. Introduction-..

Pancreatic phospholipase A 2 and non-pancreatic ascitic phospholipases A 2 were studied in sera of healthy individuals and of patients suffering from sepsis or acute pancreatitis. In gel filtration experiments, immunoreactive ascitic phospholipase A 2 , as determined in serum by a time-resolved fluoroimmunoassay, eluted either unassociated with an apparent M r of 10 000 -14 000 or associated with proteins of high molecular mass. Catalytically active ascitic phospholipase A 2 was associated with high molecular weight proteins. In acute pancreatitis the catalytically active and immunoreactive pancreatic phospholipase A 2 eluted mainly as a protein of M r of 14000. The results of the gel filtration experiments indicate that pancreatic phospholipase A 2 is not associated with other proteins in human serum, whereas ascitic phospholipase A 2 is associated with protein(s) of relative high molecular weight, or exists in different polymeric forms. We also purified phospholipase A 2 from sera of healthy individuals by ion exchange chromatography and HPLC. The enzyme was homogenous, displayed an M r of approximately 13 500 as judged by SDS-polyacrylamide gel electrophoresis, and reacted with an antibody raised against ascitic phospholipase A 2 .

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.