A monoclonal antibody specific to SHBG is immobilized on microwell plates, and another monoclonal antibody, also specific to SHBG, is conjugated with horseradish peroxidase (HRP).
SHBG from the sample is bound to the plates. After a washing step, HRP conjugate is added. After a second washing step, enzyme substrate is added.
The enzymatic reaction is proportional to the amount of SHBG in the sample. The reaction is terminated by adding stopping solution. Absorbance is measured on a plate reader.