Thyroid Stimulating Hormone (TSH) - ELISA

Thyroid Stimulating Hormone(TSH)


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 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.


Any microwell reader capable of determining at 450 nm may be used. The TSH 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 TSH 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 (uIU/ml) Absorbance 450nm TSH (uIU/ml)
A1 0
B1 0
A2 0.5
B2 0.5
A3 2.5
B3 2.5
A4 10
B4 10
A5 20
B5 20
A6 40
B6 40
A7 Patient I 0.154 2.25
B7 Patient II 0.098 1.35

1.        Buger, H.G. and Y.C. Patel, Thyropin Releasing Hormone TSH, J. Clin. Endocrinol and Metab. 6:83-100, 1977
2.        Field, J.B., Pituitary Thyrotropin: Mechanism of Action in the Thyroid, S. Werner and S.H. Ingbar. Ed. Harper & Row. Hagerstwon, MD, 1978
3.        Hamilton, C.R., L.C. Adams and F. Maloof, Hyperthyroidism Due to Thyrotropin Producing Pituitary Chromophobe Adenoma, New England J. Med. 283, 1077, 1970
4.        Hock F.L., Metabolic Effect of Thyroid Hormones, In handbook of Physiology, sec. 7: Endocrinology vol. III Thyroid M.A. Greer and D.H. Solomon, Ed. Williams and Wilkins. Baltimore, MD 1972
5.        Ingbar, S.H. and K.A. Woebar, The Thyroid Gland, in the textbook of endocrinology R.H. Williams Ed. W.BN. Suanders & Co. Philadelphia, PA 1981
6.        Lyon, I.C.T. ,Screening for Congenital Hypothyroidism, three years experience New Zealand Me. J. 97, 175, 1984
7.        Ornston B.J. ,Clinical Effects of TRH Release After i.v. and Oral Administration in Normal Volunteers and Patients with Tyroid Disease, Frontiers of Hormone Research 1, 45-75, 1972
8.        Parker, D.C. Pekary, A.E. et al, Effects of Normal and Reserved Sleepwake Cycles Upon Nychtohemeral Rhythmicity of Plasma Thryotropin: Evidence suggestive of an Inhibitory Influence in Sleep, J. Clin. Endocrinol. Metab. 43, 318. 1976
9.        Patel, Y.C. and H.G. Burger,Serum Thyrotroin (TSH) in Pituitary and/or Hypothalamic Hypothryroidism: Normal or Elevated Basal Levels and Paradoxical Responses to Thyrotropin Releasing Hormone, J. Clin Endocrinol. Metab. 37, 190. 1973
10.     Pierce, J.C. , Pituitary Thyrotropin: Chemistry in the Thyroid, S. Werner and S.H. Ingbar, Ed. Harper & Row, Hagerstown. MD, 1978
11.     Scanlon, M.F., B.R. Smith et al Thyroid-Stimulating Hormone: Neuro-regulation and Clinical Applications, Clin. Sci. Mol Med. 55 1 & 129, 1978