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.
Raverot
Véronique
V
Inserm, U863, Lyon F-69003, France. veronique.raverot@chu-lyon.fr
Lopez
Jonathan
J
Grenot
Catherine
C
Pugeat
Michel
M
Déchaud
Henri
H
D000645:Ammonium Sulfate / Q000737:chemistry; D055910:Antibodies, Immobilized / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D005260:Female; D006801:Humans; D008280:Magnetics; D008297:Male; D011485:Protein Binding; D011863:Radioimmunoassay / Q000379:methods*; D012738:Sex Hormone-Binding Globulin / Q000378:metabolism*; D013739:Testosterone / Q000097:blood*
D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D012738:Sex Hormone-Binding Globulin; D013739:Testosterone; D000645:Ammonium Sulfate