MONOCLONAL-ANTIBODIES AGAINST BOVINE ADRENAL CYTOCHROME-P-450(11-BETA) AND CYTOCHROME P-450SCC - THEIR ISOLATION, CHARACTERIZATION AND APPLICATION TO IMMUNOHISTOCHEMICAL ANALYSIS OF ADRENAL-CORTEX

  • 1 December 1985
    • journal article
    • research article
    • Vol. 23  (6A) , 1013-1021
Abstract
Seven monoclonal antibodies directed against bovine adrenocortical cytochrome P-45011.beta. were isolated. They were found to be immunoglobulins each having distinct affinity to the antigen. Five clones had IgG1 heavy chains, whereas the other two had IgG2b heavy chains. All the clones had .kappa. light chains. All the monoclonal antibodies recognized a protein among mitochondrial proteins of bovine adrenal cortex, whose mol. wt, 50,000 was the same as that of cytochrome P-45011.beta.. Among the monoclonal antibodies isolated, monoclonal antibody 258 recognized a protein of Mr 50,000 in rat adrenal mitochondria, a protein of Mr 47,000 in pig adrenocortical mitochondria, a protein of Mr 50,000 in guinea pig adrenal mitochondria, a protein of Mr 55,000 in rabbit adrenal mitochondria and a protein of Mr 50,000 in human adrenocortical mitochondria. There results suggest that each of these proteins, having a mol. wt around 50,000 recognized by monoclonal antibody 258 is very likely to be the cytochrome P-45011.beta. of adrneocortical mitochondria of each animal species. Monoclonal antibody 42718 inhibited by 50% the steroid hydroxylation activity of cytochrome P-45011.beta.. In addition to the monoclonal antibodies against cytochrome P-45011.beta., three monoclonal antibodies directed against bovine adrenocortical cytochrome P-450SCC were also isolated and characterized. Immunohistochemical staining of bovine adrenal cortex with the use of these two kinds of monoclonal antibodies revealed that the contents of both cytochrome P-45011.beta. and cytochrome P-450SCC were greater in the zona fasciculata and zona reticularis than in the zona glomerulosa. Electron microscopical observation of immunoperoxidase-stained preparations confirmed the presence of these cytochromes in the mitochondria.