1.        Before beginning the test, bring all samples and reagents to room temperature (20-25°C) and gently mix.
2.        Have all reagents and samples ready before the start of the assay. Once the test has begun it must be performed without any interruptions to get the most reliable and consistent results.
3.        Use new disposable tips for each specimen.


1.        Secure the desired number of coated wells in the holder.
2.        Dispense 50 ul of Standards, Controls or Serum samples.
3.        Dispense 100 ul of enzyme conjugate to each wells
4.        Incubate for 60 minutes at room temperature.
5.        Remove incubation mixture and rinse the wells 5 times with running tap water.
6.        Dispense 100 ul of Solution A and then 100 ul of Solution B into each well.
7.        Incubate 30 minutes at room temperature.
8.        Stop reaction by adding 50 ul of 1N H2SO4 into each well.
9.        Read O.D. at 450 nm with a microwell reader against the blank well which contain only Solution a and solution B and stopping reagent.


Any microwell reader capable of determining at 450 nm may be used. The T3 value of patient is obtained as follows:
1.        Plot the concentration (X) of each Reference Standards against its absorbance (Y) on full logarithmic graph paper.
2.        Obtain the value of patient T3 by reference to the Standard Curve. For example: (This data is for demonstration purposes only and must not be used in place of data generated for each assay).

Well No. Description (ng/dL) Absorbance 450nm T3(ng/dL)
A1 0 3.079
B1 0 3.019
A2 50 2.563
B2 50 2.557
A3 100 1.988
B3 100 1.908
A4 250 1.289
B4 250 1.349
A5 500 0.757
B5 500 0.795
A6 1000 0.43
B6 1000 0.434
A7 Patient I 2.545 45.1
B7 Patient II 1.345 229.9

1.        Beeler, M.F. Interpretation in Clinical Chemistry, ASCP. P 446-453, 1978.
2.        Surks M.I. et al. A new Radioimmunoassay for plasma triiodothyronine: measurements in thyroid disease and in patients maintained on hormonal replacement. J. Clin. Invest. 51(12), 3104, 1972

3.        Sterling K. et al. T3 Thyrotoxicosis: thyrotoxicosis due to elevated serum triiodothyronine level. J.A.M.A. 213:571-575, 1970.
4.        Burke, C.W. Eastman C.J. Thyroid hormones. Brit. Med Bull 30:93-99, 1974.
5.        Utiger R.D. Serum triiodothyronine in man. Ann Rev Med 25:289-302, 1974.
6.        Spector D.A. et al Thyroid function and metabolic state in chronic renal failure. Ann Int. Med. 85:724-730, 1976.
7.        Burr W.A. et al. Serum triiodothyronine and reverse triiodothyronine concentrations after surgical operation. Lancet II: 1277-1279, 1975.
8.        Cavalieri, R.R. and B. Rapoport. Impaired peripheral Conversion of Thyroxine to triiodothyronine. Ann Rev. Med. 28:57-65, 1977.
9.        McLarty D.G. et al.Tyroid-hormone levels and prognosis in patients with serious non-thyroid illness. Lancet II. 275-276, 1975.