DBC Thyroid Stimulating Hormone (TSH) Elisa

PRINCIPLE OF THE TEST 
The principle of the following enzyme immunoassay test follows a typical one-step capture or ‘sandwich’ type assay. The assay makes use of two highly specifi c monoclonal antibodies: A monoclonal antibody specifi c for TSH is immobilized onto the microplate and another monoclonal antibody specifi c for a different region of TSH is conjugated to horse radish peroxidase (HRP). TSH from the sample and standards are allowed to bind simultaneously to the plate and to the HRP conjugate. The washing and decanting steps remove any unbound HRP conjugate. 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 by the enzymatic reaction is directly proportional to the concentration of TSH in the sample. 
A set of standards is used to plot a standard curve from which the amount of TSH in patient samples and controls can be directly read.

CLINICAL APPLICATIONS 
Thyroid stimulating hormone (TSH) is a glycoprotein hormone secreted by the anterior pituitary gland. TSH has two subunits, namely α and β. The α subunit of TSH is similar to the α subunit found in the LH, FSH and hCG glycoprotein hormones. The β subunit however, is specifi c and differs from hormone to hormone. The thyroid hormones are secreted and produced by the thyroid gland. The production of thyroid hormones is under the regulation of TSH. Also, TSH acts as a stimulator of iodide transport and the gland itself is under the positive control of TSH. The concentrations of thyroid hormones control the secretion of TSH, therefore, a negative feedback exists. It is to be noted that the secretion of thyroid hormones are under the direct, positive effect of the sympathetic nervous system. 
The major protein component of the thyroid gland is thyroglobulin, a glycoprotein of which the secretion in the blood stream is stimulated by TSH. Therefore, TSH plays an important role in the proper function and development of the thyroid gland.

SPECIMEN COLLECTION AND STORAGE 
Approximately 0.2 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 30 μIU/mL, then dilute it with calibrator A at a dilution of no more than 1:8. The result obtained should be multiplied by the dilution factor.