DBC Sex Hormone Binding Globulin (SHBG) Elisa

PRINCIPLE OF THE TEST 
The principle of the following enzyme immunoassay test follows a typical two-step capture or ‘sandwich’ type assay. The assay makes use of two highly specifi c monoclonal antibodies: A monoclonal antibody specifi c for SHBG is immobilized onto the microplate and another monoclonal antibody specifi c for a different region of SHBG is conjugated to horse radish peroxidase (HRP). SHBG from the sample and standards are allowed to bind to the plate, washed, and subsequently incubated with the HRP conjugate. After a second washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of the stopping solution. The absorbance is measured on a microtiter plate reader. The intensity of the colour formed by the enzymatic reaction is directly proportional to the concentration of SHBG in the sample. 
A set of standards is used to plot a standard curve from which the amount of SHBG in patient samples and controls can be directly read.

CLINICAL APPLICATIONS 
Sex hormone binding globulin (SHBG) is a glycoprotein composed of 373 amino acid residues and three carbohydrate side chains. SHBG has been known by many other names including Testosteroneestradiol Binding Globulin (TeBG), Sex steroid Binding Protein (sBP) and Sex Steroid Binding Globulin (SSBG). One of the main properties of SHBG is its high affi nity for steroids, especially the C18, C19 and 17α-hydroxyl groups. The binding of steroids to SHBG is temperature and pH dependent. The three steroids that have a high avidity for SHBG are Dihydrotestosterone, Testosterone and Estradiol. Very small amounts of these steroids are free in biological fluid; the majority are bound to SHBG and albumin. These two fractions, that is, free and bound exist in a state of dynamic equilibrium. When the level of SHBG concentration changes, a remarkable change occurs in both albumin-bound hormone and also in the free fraction.
Throughout life SHBG increases until the eighties in both sexes. During the menstrual cycle SHBG does not seem to vary appreciably, however, according to some authors the concentration of SHBG is elevated in the luteal phase. During pregnancy the level of SHBG rises rapidly until about the 30th week of gestation.

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 4°C for up to 24 hours or at -10°C or lower if the analyses are to be done at a later date. Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling.

CALCULATIONS 
1. Calculate the mean optical density of each calibrator duplicate. 
2. Calculate the mean optical density of each unknown duplicate. 
3. Subtract the mean absorbance value of the “0” calibrator from the mean absorbance values of the calibrators, controls and serum samples. 
4. Draw a calibrator curve on log-log paper with the mean optical densities on the Y-axis and the calibrator concentrations on the X-axis. If immunoassay software is being used, a 4-parameter or 5-parameter curve is recommended. 
5. Read the values of the unknowns directly off the calibrator curve. 
6. If a sample reads more than 295 nmol/L, then dilute it with assay buffer at a dilution of no more than 1:10 (from original 1:10 dilution). The result obtained should be multiplied by the dilution factor.