Alternative ELISA for Sex Hormone-Binding Globulin in Plasma

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Abstract
Bioavailable testosterone is often derived from the free androgen index (1) , which is reliant on measurements of both testosterone and sex hormone-binding globulin (SHBG). Although testosterone assays are widely available in various formats, assays for SHBG are more restricted partly because of the absence of automated procedures and the relatively high expense of kits. Dako (Denmark) has a rabbit polyclonal antibody to SHBG as well as, until recently, a peroxidase-labeled rabbit polyclonal antibody to SHBG. These two antibodies can be used together with pooled human pregnancy plasma as a calibrator to provide a direct inexpensive ELISA for SHBG in plasma, as detailed by Dako in their package insert and their “general ELISA procedure” guide. Briefly, microtiter plates are coated with antibody diluted 1:350 in bicarbonate buffer overnight at 4 °C, followed by “blocking” and the addition of either the calibrator (diluted pregnancy plasma) or diluted plasma samples for an overnight incubation at 4 °C. The next day, the plate is washed, and bound SHBG is detected with a 6-h incubation using the polyclonal peroxidase-labeled SHBG antibody (1:3000 dilution). The plates are finally washed, and o-phenylenediamine substrate is added.

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