Arachidonate 5-lipoxygenase of porcine leukocytes studied by enzyme immunoassay using monoclonal antibodies.

The cytosol fraction of porcine leukocytes contained 5-lipoxygenase, the activity of which was masked by a predominant activity of 12-lipoxygenase. The 5-lipoxygenase was partially purified to a specific activity of about 10 nmol of arachidonic acid oxygenated/min/mg of protein and given to mice as an antigen to prepare monoclonal antibodies against the enzyme. Two species of antibodies recognized separate sites of the 5-lipoxygenase protein and did not cross-react with 12-lipoxygenase. They were utilized to develop a peroxidase-linked immunoassay of sandwich-type, which allowed a quantitative determination of the 5-lipoxygenase protein. The assay was applied to a screening of the 5-lipoxygenase content in various porcine tissues. By far the highest content of 5-lipoxygenase was found in leukocytes. About one-tenth the amount of the enzyme was found in lung, pancreas, ileum, and thymus, which could not be attributed to the contaminating leukocytes in these tissues.