Inserm, U863, Lyon F-69003, France.
Anal Chim Acta. 2010 Jan 18;658(1):87-90. doi: 10.1016/j.aca.2009.10.057. Epub 2009 Oct 29.
Testosterone (T) circulates in the blood tightly bound to sex hormone-binding globulin (SHBG) and weakly to albumin. Measuring protein unbound T (free) or non-SHBG-bound T rather than total T has been recommended for the evaluation of androgen disorders in humans. Ammonium sulfate precipitation has been widely used to separate [SHBG-T] complex from free and albumin-bound T. To achieve more specificity in this separation, we used monoclonal anti-SHBG antibody and developed a suitable and convenient immunoassay for measuring non-SHBG-bound T. Magnetic beads were covalently coupled to a monoclonal anti-SHBG antibody to capture [SHBG-T] complex from plasma samples. Magnetic separation was then performed to allow measurement of non-SHBG-bound T in the supernatant by direct radioimmunoassay. When 300 microL of plasma samples were incubated at room temperature with 10 microL of anti-SHBG beads, residual SHBG concentration was undetectable in the supernatant. The specificity of proteins retained on anti-SHBG beads was further demonstrated by peptide mass fingerprint on a MALDI-TOF analyzer. The non-specific adsorption of T on beads was low (5%), and dissociation of T from SHBG-T complex was less than 5% after 180 min of incubation. The plasma concentrations of non-SHBG-bound T using anti-SHBG beads were highly correlated to those obtained using ammonium sulfate precipitation. We conclude that SHBG immunocapture is a highly specific and useful tool for an experimental direct measurement of plasma non-SHBG-bound T. This methodology is also convenient and appropriate for routine and automated assay.
睾酮（T）在血液中与性激素结合球蛋白（SHBG）紧密结合，与白蛋白弱结合。有人建议，为了评估人类的雄激素紊乱，应测量未结合蛋白的 T（游离）或非 SHBG 结合的 T，而不是总 T。硫酸铵沉淀已广泛用于从游离 T 和白蛋白结合的 T 中分离 [SHBG-T] 复合物。为了在这种分离中实现更高的特异性，我们使用了单克隆抗 SHBG 抗体，并开发了一种合适且方便的免疫测定法来测量非 SHBG 结合的 T。将磁珠共价偶联到单克隆抗 SHBG 抗体上，从血浆样品中捕获 [SHBG-T] 复合物。然后进行磁分离，以便通过直接放射免疫测定法测量上清液中非 SHBG 结合的 T。当 300 μL 的血浆样品在室温下与 10 μL 的抗 SHBG 珠孵育时，上清液中无法检测到残留的 SHBG 浓度。通过 MALDI-TOF 分析仪上的肽质量指纹图谱进一步证明了保留在抗 SHBG 珠上的蛋白质的特异性。T 在珠上的非特异性吸附率较低（5%），并且在孵育 180 分钟后，T 从 SHBG-T 复合物中的解离率小于 5%。使用抗 SHBG 珠获得的非 SHBG 结合的 T 的血浆浓度与使用硫酸铵沉淀获得的浓度高度相关。我们得出结论，SHBG 免疫捕获是一种非常特异且有用的工具，可用于实验性地直接测量血浆中非 SHBG 结合的 T。该方法还方便且适合常规和自动化测定。
