Quantitative Analysis of Total Tyroxine (T4) concentration in human serum.

Introduction Explanation of the Test

Thyroid gland is the source of L- thyroxine (T4), Where (T4) is synthesized and stored. Thyroxine is one type of hormone, which circulates in the blood as an equilibrrium mixture of free and serum bound hormone. More than 99% of T4 is revesibly bound to three plasma proteins in blood – thyroxine binding globulin(TBG) 70%, albumin (10%) and thyroxine binding pre-albumin(TBPA) (20 %). Less than 0.03% T4 is present in the circulation as unbound state in the blood. This small T4 amount represents the physiologically available hormone which is biologically active.

The presence of thyoid disorders in patients is determined by Immunoassay test. This test is the most reliable convenient and fast screening test for the determination of total T4. Levels of T4 are found to increase in hyper-thyroidism due to Grave’s disease and Plummer’s disease and in acute and subacute thyroiditis. Low levels of T4 have been linked with congenital hypothyroidism, myxedema, chronic thyroiditis (Hashimoto’s disease), and with some genetic abnormalities.

Principle of the Test

In this T4 EIA, Some amount of anti-T4 antibody is coated on microtiter wells. A constant amount of T4 conjugated with Alkaline phosphatase and a measured amount of human serum are added to the micro titer wells and sake before incubation. During incubation , T4 and conjugated T4 compete for the limited binding sites on the anti T4 antibody. After 30 minutes incubation at 370C temperature, the wells are washed by Tris buffer 4 times to removed unbound T4 conjugate. A constant amount of chemiluminescent Reagent is then added in each well and sake well and keep the plate in the sample chamber of the luminometer and measure the intensity of l8ght (in RLU) after every 15 minutes at 450 nm. The intensity of the chemiluminescent light is proportional to the amount of enzyme present and is inversely proportional to the amount of unlabeled T4 in the sample. T4 standards is tested in the same way as reference, and then calculate the T4 concentration in the unknown sample.


Materials provided in the kit: -

1 - Rabbit anti-T4 coated microtiter wells, 96 wells.

2 - T4 Reference Standars: 0.05, 0.1, 0.5, 1.5, 5.0, 10.0, 25.0, 50.0 mg/dl, 1 set. 2.0 ml, ready to use.

3 - Alkaline Phosphatase Enzyme conjugate concentrate (10 X), 1.0 ml

4 - Enzyme conjugate Diluent, 9 ml.

5 - Washing Buffer Concentrate(10 X), 20 ml.

6 - Chemiluminescent Reagent, 15 ml

Material required but not provided:-

1 - Precision pipettes: 50ml, 100ml and 1.0 ml.

2 - Disposable pipette tips.

3 - Vortex mixture or equivalent.

4 - Paper towel absorbent paper.

5 - Luminometer.

Specimen Collection and Reagent Preparation

Whole blood sample should be taken by appropriate medical techniques and make the serum sample for testing. This kit is for use with serum samples without additives.

Reagent Preparation

1 - All reagents should be warm up to room temperature (20-250C) before use.

2 - Preparation of working T4-Alkaline Phosphatase Conjugate Reagent: 1.0 ml of T4-AP conjugate concentrate (10X) mix to 9.0 ml of T4 Conjugate Diluent (1:9 dilution) and mix well.

3 - Preparation of Washing Buffer from Washing Buffer Concentrate: 1 ml of Washing Buffer Concentrate (10X) mix to 9 ml of DI water(1:9dilution), and mix well.

Assay Procedure

1 - Place the required number of wells in to the well holder.

2 - Pipette the 50ml of washing buffer, T4 standards, samples and controls add in to the degignated wells.

3 - Pipette the 75ml of working conjugate reagent and place in to each well.

4 - Shake the plate on vibrator or on appropriate mixer for 20-30 seconds.

5 - Incubate the plate in the oven at 370C for 30 minutes.

6 - Take out the plate from oven and remove the mixture from the wells by flicking the plate into a waste container.

7 - Wash the wells four times with dilute Wash buffer(do not use any other buffer).

8 - Dry the wells containing plate on paper towel by striking.

9 - Now add the 125ml Chemiluminescent Reagent into each well and shake the plate on the vibrator for at least 10 seconds.

10 - Keep the plate inside the sample chamber of luminometer. And start taking the reading after every 15 minutes interval.

Calculation of the Results

1 - Take out the average intensity values(in RLU) for each set of reference standards, control and samples.

2 - Plot a standard curve with average intensitiy (in RLU) for each reference standard with its concentration in ml/dl on the graph paper, Plot the Intensity (in RLU) on the Y axis and concentration of the standards on X axis.

3 - Calucate the average value of intensity (in RLU) for each sample, determine the corresponding concentration of T4 in ml/dl from the standard curve.

Example of a Standard Curve

Results of a typical standard run with Intensity of light (in RLU) shown in the Y axis against T4 concentrations shown in the X axis. This standard curve is for the purpose of illustration only, each usershould obtain his or her own data and standard curve.

Sr.No.Conc. Of T4 in ug/dlIntensity in RLU in 15 min
1 -01782322
2 - 0.051320881
3 - 0.11082446
4 - 0.5680037
5 - 1.5522852
6 - 5282359
7 - 10144392
8 - 2548559
9 - 5043430


Limitations of the Procedure

1 - Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the package insert instructions and adherence to good laboratory practice.

2 - The wash procedure is critical, insufficient washing will give poor precision and falsely elevated intensity readings and higher bacground.

3 - Incubation time and temperature are both critical. Follow the written instruction in the insert.