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 25 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 contains only Solution A , Solution b and stopping reagent.


Any microwell reader capable of determining at 450 nm may be used. The T4 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 T4 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 (ug/dL) Absorbance 450nm T4 (ug/dL)
A1 0 3.079
B1 0 3.019
A2 1.5 2.65
B2 1.5 2.666
A3 3 2.101
B3 3 2.285
A4 6 1.754
B4 6 1.754
A5 12 1.25
B5 12 1.18
A6 24 0.772
B6 24 0.778
A7 Patient I 1.982 4.4
B7 Patient II 0.987 16.6

1.        Beeler, M.F. Interpretation in Clinical Chemistry, ASCP. P446-453, 1978.
2.        Oppenheimer, J.H. Role of Plasma Proteins in the binding, distribution and metabolism of the thyroid hormones. New Engl. J. Med. 278:1153-1162.
3.        Schall R., Fraser, A., Hansen, H., Kern, C., and Tenoxo, H.: Sensitive Manual Enzyme Immunoassay for Thyroxine. Clin Chem 24(10), 1978
4.        Sharpe, S., Coorman, W., Blomme, W., and Lackeman, G. Quantitative enzyme Immunoassay Current Status. Clin Chem 22(6), 1976.
5.        Wilson, G. Enzyme Immunoassay. Clin. Chem 22(8), 1976
6.        Schuurs, A. and Van Weemen, B. Review, Enzyme Immunoassay. Clin Chem Acta, 81(5), 1977