DBC Dihydrotestosterone (DHT) ELISA

PRINCIPLE OF THE TEST 
The DHT ELISA is a competitive immunoassay. Competition occurs between DHT present in calibrators, controls and patient samples and an enzyme-labelled antigen (conjugate) for a limited number of anti-DHT antibody binding sites on the microplate wells. After a washing step that removes unbound materials, the enzyme substrate is added, and 30 minutes later the enzymatic reaction is terminated by addition of stopping solution. The resulting optical density (OD), measured with a microplate reader, is inversely proportional to the concentration of DHT in the sample. A calibrator curve is plotted with a provided set of calibrators to directly calculate the concentration of DHT in patient samples and controls.

CLINICAL APPLICATIONS 
Dihydrotestosterone (DHT) is the most active natural androgen in humans with a principal role in the development of primary and secondary sexual characteristics and potential participation in a myriad of other physiological processes. The bulk of androgen production takes place mainly in the Leydig cells of the testes. Androgens circulate in the blood bound to proteins, especially sex hormone binding globulin (SHBG) from peripheral conversion of testosterone, while in females most of the DHT is derived from androstenedione. Some of the main clinical indications of the DHT measurement in serum are investigations of delayed puberty in men and evaluation of the presence of active testicular tissue.

SPECIMEN COLLECTION AND STORAGE 
Approximately 0.1 mL of serum is required per duplicate determination. Collect 4–5 mL of blood into an appropriately labelled tube and allow it to clot. Centrifuge and carefully remove the serum layer. Store at room temperature for up to seven days, at 2–8°C for up to fourteen days or freeze at or below -20°C for up to 1 month. Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling.

CALCULATIONS 
1. Calculate the mean optical density of each calibrator, control and specimen sample. 
2. Use a 4-parameter or 5-parameter curve fit with immunoassay software to generate a calibrator curve. 
3. Read the values of the unknowns directly off the calibrator curve. 
4. If a sample reads more than 2,500 pg/mL, refer to Limitations section, point 4.