Characterization of a monoclonal antibody to hog thyroid peroxidase and its use for immunohistochemical localization of the peroxidase in the thyroid gland.
A monoclonal antibody (30.1.2) to hog thyroid peroxidase was produced, purified, and characterized. The IgG of 30.1.2 formed an immune complex with the peroxidase in a 1:2 or 1:1 molar ratio depending on the IgG to antigen ratio in the incubation mixture. Immune complex formation did not inhibit the peroxidase activity, which was actually activated 2-fold in the 1:1 complex. Studies of the binding of the conjugate of the IgG or its Fab' with horseradish peroxidase to untreated and acetone-treated thyroid microsomes showed that the IgG conjugate could bind to only a very small portion of the total binding sites (thyroid peroxidase) present in untreated microsomes even after prolonged incubation. The binding of the Fab' conjugate to untreated microsomes, on the other hand, increased as the incubation time was increased, reaching 40% of the total sites after 20 h of incubation. These findings indicated that thyroid peroxidase is localized on the inner surface of the microsomal membranes and that the Fab' conjugate, but not the IgG conjugate, can slowly penetrate through the membrane barrier to reach the peroxidase. Immunohistochemical experiments using the Fab' conjugate as a probe revealed that most thyroid peroxidase in the thyroid gland is located in the endoplasmic reticulum and perinuclear cisternae of the follicular cell, although a small amount could occasionally be detected in the apical membrane including microvilli. In contrast to previous reports, no thyroid peroxidase could be found in other cellular structures such as Golgi apparatus and apical vesicles by the immunohistochemical technique employed.
D000818:Animals; D000911:Antibodies, Monoclonal; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006651:Histocytochemistry; D007124:Immunoenzyme Techniques; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007453:Iodide Peroxidase / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology; D010544:Peroxidases / Q000378:metabolism*; D013552:Swine; D013961:Thyroid Gland / Q000201:enzymology* / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D010544:Peroxidases; D007453:Iodide Peroxidase