Use of non-radioactive labels for half-life measurement of sex hormone-binding globulin in the rabbit.
The purpose of this study was to investigate two methods for labeling rabbit sex hormone-binding globulin (rSHBG) with non-radioactive material, biotin (B) and europium (Eu3+), in order to obtain stable labeled SHBG and measure in vivo its metabolism and distribution. The obtained half-life values were compared with [125I]rSHBG half-lives. rSHBG was first isolated by immunoaffinity chromatography using an immobilized monoclonal anti-human SHBG (hSHBG) antibody that cross-reacts with rSHBG. This purified rSHBG was labeled by either biotin-X-N-hydroxysuccinimide ester (rSHBG-B), Eu3(+)-diethylenetriaminepentaacetic dianhydride, or Eu(3+)-isothiocyanatobenzyldiethylenetriamine-tetraacetic acid reagents (rSHBG-Eu3+) or by 125I using Bolton and Hunter reagent ([125I]rSHBG). The labeling procedure preserved the main properties of native SHBG: interaction with the lectine concanavaline A-Sepharose, recognition by anti-hSHBG monoclonal antibody, and, although lower than in native SHBG, the binding affinity for 5 alpha-dihydrotestosterone. These characteristics were the prerequisite for reliable measurement of the metabolism of labeled SHBG. Labeled rSHBG was injected into various rabbits with blood sampling at 2 min and at 1, 2, 4, 8, 12, 24, 48, 72, and 96 h after injection. rSHBG-B or desiaylated rSHBG-B and rSHBG-Eu3+ were captured from serum samples by tubes coated with anti-hSHBG antibody prior to the following detection procedure: biotin was detected by luminometry with the [streptavidin-alkaline phosphatase-dioxetane (AMPPD)] system and europium by time-resolved fluorimetry. [125I]rSHBG was detected by measurement of radioactivity either directly on serum or after fixation on concanavaline A-Sepharose.(ABSTRACT TRUNCATED AT 250 WORDS)
Fimbel
S
S
Laboratoire de la Clinique Endocrinologique, Hôpital de l'Antiquaille, Lyon, France.
Déchaud
H
H
Grenot
C
C
Tabard
L
L
Claustrat
F
F
Bador
R
R
Pugeat
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D001710:Biotin; D002846:Chromatography, Affinity; D013196:Dihydrotestosterone / Q000378:metabolism; D005063:Europium; D006207:Half-Life; D007457:Iodine Radioisotopes; D007700:Kinetics; D008297:Male; D011817:Rabbits; D012738:Sex Hormone-Binding Globulin / Q000032:analysis* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007457:Iodine Radioisotopes; D012738:Sex Hormone-Binding Globulin; D013196:Dihydrotestosterone; D005063:Europium; D001710:Biotin