Monoclonal antibodies to thromboxane synthase from porcine lung. Production and application to development of a tandem immunoradiometric assay.
Two hybridoma cell lines secreting antibodies against thromboxane synthase of porcine lung were produced. Clone TS1 secretes IgG2a antibody of lower affinity, while clone TS2 secretes IgG1 antibody of higher affinity. Both antibodies (when bound to rabbit anti-mouse IgG-Staphylococcus aureus complex) can immunoprecipitate thromboxane synthase from crude enzyme preparations in an active form suggesting that binding was not directed at the active site. Each antibody showed a distinctive pattern of cross-reactivity with thromboxane synthase from different porcine tissues. Neither of the antibodies cross-reacted with the enzyme from tissues of other species tested, indicating the heterogeneous nature of the enzyme among species. Competitive binding assay revealed that TS1 and TS2 recognized different determinants on the enzyme. The fact that two antibodies bind to separate epitopes on the same enzyme allows the development of a sensitive tandem immunoradiometric assay. The assay, based on binding of 125I-TS2 to thromboxane synthase immobilized on TS1-S. aureus complex, was linear with 7.5 approximately 75 ng of purified lung thromboxane synthase as standards and applicable to enzyme preparations regardless of their purity. The concentration of immunoreactive thromboxane synthase in porcine tissues as determined by this assay followed the order of platelet greater than colon greater than duodenum greater than lung greater than kidney greater than stomach. The fact that gastrointestinal tract is enriched with thromboxane synthase suggests that thromboxane may have significant physiological roles to be recognized in these organs.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001667:Binding, Competitive; D003429:Cross Reactions; D006801:Humans; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D009928:Organ Specificity; D011863:Radioimmunoassay / Q000379:methods; D013045:Species Specificity; D013552:Swine; D013930:Thromboxane-A Synthase / Q000032:analysis* / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D013930:Thromboxane-A Synthase