Solid-phase iodothyronine-5'-deiodinase (5'-D) assays applied in production of monoclonal antibodies against 5'-D
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Solid-phase iodothyronine-5'-deiodinase (5'-D) assays applied in production of monoclonal antibodies against 5'-D. / Boye, N.; Frokiaer, H.; Kaltoft, K.; Laurberg, P.
I: Journal of Endocrinology, Bind 118, Nr. 3, 1988, s. 439-445.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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T1 - Solid-phase iodothyronine-5'-deiodinase (5'-D) assays applied in production of monoclonal antibodies against 5'-D
AU - Boye, N.
AU - Frokiaer, H.
AU - Kaltoft, K.
AU - Laurberg, P.
PY - 1988
Y1 - 1988
N2 - 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/l 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 enzyme-binding 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 activity in various tissues.
AB - 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/l 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 enzyme-binding 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 activity in various tissues.
UR - http://www.scopus.com/inward/record.url?scp=0023722560&partnerID=8YFLogxK
U2 - 10.1677/joe.0.1180439
DO - 10.1677/joe.0.1180439
M3 - Journal article
C2 - 3053961
AN - SCOPUS:0023722560
VL - 118
SP - 439
EP - 445
JO - Journal of Endocrinology
JF - Journal of Endocrinology
SN - 0022-0795
IS - 3
ER -
ID: 316996922