New approach for measurement of non-SHBG-bound testosterone in human plasma.

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.