Alternative ELISA for sex hormone-binding globulin in plasma.

To the Editor: 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. Recently this peroxidase-labeled polyclonal antibody has become unavailable, requiring the use of alternatives. We have obtained a mouse monoclonal antibody to SHBG, and its use in a modified ELISA shows excellent agreement to the Dako method (Fig. 1). The modification uses microtiter plates coated with Dako SHBG polyclonal diluted (1: 700) in phosphate-buffered saline (PBS) and identical initial incubation conditions. The method differs in that the bound SHBG is detected using this mouse monoclonal SHBG antibody supernatant diluted 1:20 in PBS containing 1 mL/L Tween 20 and 1 g/L gelatin (assay buffer) for 30 min at room temperature, followed by washing and the addition of anti-mouse Ig peroxidase from Amersham (1:1000 in assay buffer) for an additional 30-min incubation at room temperature. The plate is finally washed, and o-phenylenediamine substrate is added. The modification described uses “assay buffer” as a common reagent for all steps except coating, which is carried out in PBS with no additives. The method offers a shorter and immediate alternative to those laboratories currently determining SHBG by ELISA using both Dako antibodies.