USE OF NONRADIOACTIVE 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 [I-125]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 Eu3+-isothiocyanatobenzyldiethylenetriamine-tetraacetic acid reagents (rSHBG-Eu3+) or by I-125 using Bolton and Hunter reagent ([I-125]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. rSHBC-B or desialylated 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. [I-125]rSHBG was detected by measurement of radioactivity either directly on serum or after fixation on concanavaline A-Sepharose. The half-life values obtained with rSHBG-B (t(1/2)alpha = 5.5 +/- 0.6 h; t(1/2)beta = 34.8 +/- 5.0 h; n = 4) were comparable to those obtained with [I-125]rSHBG (t(1/2)alpha = 4.5 and 4.0 h; t(1/2)beta = 36.0 and 37.0 h). As previously described for many glycoproteins, treatment of rSHBG-B by neuraminidase induced a dramatic decrease in the half-life of this asialo-rSHBG preparation (t(1/2)alpha = 7.0 and 8.0 min; t(1/2)beta = 4.0 and 4.0 h). The shouter half-life observed with rSHBG-Eu3+ could be explained by the release of europium from the chelates in vitro and probably in vivo. In conclusion, biotinylated SHBG is a stable protein with the main characteristics of native SHBG and provides greater detection sensitivity when coupled to a streptavidin-alkaline phosphatase-AMPPD chemiluminescent detection system. The labeling with biotin should be a useful tool for investigating metabolism and specific uptake in target tissue of various forms of recombinant SHBG.