Studies on porcine arachidonate 12-lipoxygenase using its monoclonal antibodies

Abstract

Monoclonal antibodies were raised against arachidonate 12-lipoxygenase using a partially purified enzyme from porcine leukocytes as an antigen. Immunohistochemical studies indicated a selective localization of 12-lipoxygenase in the cytosol of polymorphonuclear leukocytes. Two separate species of antibody (lox-1 and lox-2) recognizing different sites of the enzyme protein were utilized to develop a method to determine the amount of 12-lipoxygenase protein rather than the activity of enzyme. Fab fragment of lox-2 was conjugated to horseradish peroxidase, and the conjugate as a label was bound to 12-lipoxygenase. The complex was precipitated with the aid of the other antibody (lox-1) and protein A, and the peroxidase activity in the precipitate was correlated with the amount of 12-lipoxygenase. The immunoenzymometry of 12-lipoxygenase was more convenient and sensitive than the conventional assay to determine the conversion of [1-14C]arachidonic acid. Furthermore, in several porcine tissues this method allowed quantitation of the enzyme, the activity of which was masked due to the presence of certain endogenous inhibitors. A ubiquitous distribution of 12-lipoxygenase in porcine tissues was demonstrated by application of this method.