DBC Free Thyroxine (fT4 Free) Elisa

PRINCIPLE OF THE TEST 
The principle of the following enzyme immunoassay test follows the typical competitive binding scenario. Competition occurs between an unlabelled antigen (present in standards, controls and patient samples) and an enzyme-labelled antigen (conjugate) for a limited number of antibody binding sites on the microplate. The washing and decanting procedures remove unbound materials. After the 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 is inversely proportional to the concentration of fT4 in the sample. A set of standards is used to plot a standard curve from which the amount of fT4 in patient samples and controls can be directly read. 
The labelled T4 (conjugate) employed in this assay system has shown no binding properties towards thyroxine-binding globulin (TBG) and human serum albumin (HSA). The binding sites on the microplates are designed to be of a low binding-capacity in order not to disturb the equilibrium between T4 and its carrying proteins. The assay is carried out under normal physiological conditions of pH, temperature and ionic strength.

CLINICAL APPLICATIONS 
Thyroxine (T4), the principal thyroid hormone, circulates in blood almost completely bound to carrier proteins. However, only the free (unbound) fraction of thyroxine is considered to be biologically active. The main carriers of thyroxine are thyroxine-binding globulin (TBG), pre-albumin and albumin. The measurement of free thyroxine (fT4) levels correlate better with the clinical status than total thyroxine levels. 
The DBC free T4 assay is a one step competitive ELISA system that is rapid and easy to perform compared to equilibrium dialysis and ultrafi ltration methods, which are cumbersome and time-consuming. This system employs a highly specifi c monoclonal antibody and a non-analog tracer that was proved experimentally to have no signifi cant binding to TBG and albumin. In the euthyroid, normal population the free T4 concentration is 7–22 pg/mL. The level of free T4 is decreased in hypothyroidism while in thyrotoxic patients the level of free T4 is increased.

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. Draw a calibrator curve on semi-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. 
3. Calculate the mean optical density of each unknown duplicate. 
4. Read the values of the unknowns directly off the calibrator curve.