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Characterization of iodothyronine-deiodinating enzymes has been difficult due to loss of enzyme activity during purification. To obtain a new tool for studying these enzymes we investigated the possibility of developing monoclonal antibodies (MAbs) against iodothyronine-5'-deiodinase (5'-D). Two specific and sensitive solid-phase microassays were developed for screening hybridoma supernatants for the presence of antibodies inhibiting rat kidney 5'-D. and antibodies binding to but not inhibiting the enzyme.

BALB/c mice were immunized with a 3-((3-cholamidopropyl) -dimethylammonio) -1- propanesulphonate (CHAPS)-solubilized 5'-D-rich membrane preparation from rat kidney cortical tissue. Spleen cells were fused with NSI-Ag 4/1 mouse myeloma cells by means of polyethylene glycol.

Two hybridoma cell lines (AF5 and BE8) secreting MAbs specifically binding to without inhibiting 5'-D were produced. The AF5 antibody was of the IgG_2a subclass and the BE8 antibody of the IgG_2b subclass. Binding of one of the antibodies to the enzyme inhibited binding of the other in both an enzyme-linked immunosorbent assay (ELISA) and a specific enzymebinding assay. CHAPS-solubilized kidney microsomal fraction was chromatographed on a Sepharose 6B column. Elution profiles of 5'-D activity and MAb-binding antigens, as measured by ELISA with both AF5 and BE8, were identical.

Monoclonal antibodies should be valuable probes in the further elucidation of the nature of the iodothyronine-deiodinating activity in various tissues.

J. Endocr. (1988) 118, 439–445