Monoclonal antibodies to hydroxyindole O-methyltransferase from bovine pineal gland

Abstract

Hybridomas that produce monoclonal antibodies to bovine hydroxyindole O-methyltransferase have been established by immunizing a mouse with hydroxyindole O-methyltransferase partially purified from bovine pineal glands followed by fusion of the spleen cells with myeloma NS-1 cells. Twenty-three clones have been isolated by screening the medium by two different methods. These clones produced various antibodies with different binding properties. Most of the antibodies belonged to IgG1 subtype, while 7 clones produced IgG2α or IgG2β antibodies. Three antibodies inhibited enzymatic activity, whereas others did not. Antibodies of 7 clones recognized enzyme protein denatured with sodium dodecyl sulfate, while other antibodies reacted only with native enzyme protein. Thus a variety of monoclonal antibodies will offer us a good tool for immunohistochemical localization of the enzyme, immunoaffinity purification of hydroxyindole O-methyltransferase, and isolation of the cDNA clones encoding it. We also report here on the immunohistochemical demonstration of hydroxyindole O-methyltransferase in bovine pineal gland and a new immunoaffinity procedure to purify the enzyme to homogeneity by use of monoclonal antibodies.