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MATERIALS PROVIDED 2. 3. Thyroid Stimulating Hormone (TSH) Catalog No. MYM/US-TSH 4. 5. 6. For In Vitro Diagnostic Use Only NAME AND INTENDED USE The MYM TSH Quantitative MYM/US-TSH is a solid phase enzyme linked immunsorbent assay. This test provides quantitative measurement of thyroid stimulating hormones (TSH) in human serum. (For in vitro diagnostic use) 7. 8. Microwell Strips: Anti-TSH antibodies coated wells, 8 x 12 strips, 96 wells Sample Diluent for sample dilution only (11 mL) Enzyme Conjugate (11 ml): Anti-TSH antibody conjugated to horseradish peroxidase Solution A (11 mL): containing H2O2. Solution B (11 mL): Tetramethybenzidine. Reference Standard Set (0.75 mL each vial): Calibrated to 0, 1.0, 5.0, 10, 20, and 40 uIU/mL in the sample diluent against with WHO 2nd IRP 80/558. Stop Solution: 2 N HCl. Well Holder: For securing individual wells. 1. 2. Microwell Reader Pipetor with tips for 50 and 100 uL STORAGE AND STABILITY 1. 2. 3. The MYM/US-TSH Quantitative is a solid phase enzyme-linked immunosorbent assay (ELISA). The wells are coated with anti-TSH antibodies. The samples, Standards and Controls are incubated in the wells with enzyme conjugate which is another antibody directed toward a different region of TSH molecules and chemically conjugated with horseradish peroxidase. Unbound enzyme conjugate is washed off and the amount of bound peroxidase is proportional to the concentration of the TSH present in the Samples, Standards and Controls. Upon addition of the substrate and chromogen, the intensity of color developed is proportional to the concentration of TSH in the serum. Store the kit at 2-8C in a refrigerator. Keep microwells sealed in a dry bag with desiccants. The unopened reagents are stable until expiration of the kit. Solution A and Solution B should be colorless; if solutions turn blue, it must be replaced. Do not expose test reagents to strong light during storage or usage. 1. 2. 3. 1. 2. 3. 4. 5. 6. WARNING AND PRECAUTION 2. 3. MYM TSH is designed for in vitro use only. The components in this kit are intended for use as an integral unit. The components of different lots should not be mixed. Handle all reference standards and test samples as potentially infectious agents. The standards were found negative for Hepatitis B, C and HIV antibodies. However, because no test methods offer complete assurance of the absence of the HIV I/II, Hepatitis B Hepatitis C virus or other infectious agents, these materials should be handled at the Biosafety Level 2 as recommended for any potentially infectious serum or blood specimen in the Centers for Disease Control/National Institutes of Health Manual” Biosafety in Microbiological and Description (uIU/ml) 0 1.0 5.0 10 20 40 Pat #1 Pat#2 Pat#3 Absorbance 450 nm 0.002 0.142 0.526 1.011 1.614 3.140 0.083 0.264 1.700 TSH (uIU/ml) 0.53 2.12 19.82 PREPARATION FOR ASSAY Before beginning the test, bring all samples and reagents to room temperature (20-25C) and gently mix. 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. Use new disposable tips for each specimen. ASSAY PROCEDURE 1. PRINCIPLE OF THE ASSAY Collect blood by venipuncture, allow to clot and separate the serum by centrifugation at room temperature. If sera cannot be assayed immediately, they can be stored at 2-8C for a week or frozen for up to six months. Avoid repeated freezing and thawing of serum sample. MATERIALS REQUIRED BUT NOT PROVIDED SUMMARY AND EXPLANATION OF TEST The secretion of TSH from anterior pituitary is controlled by thyrotropin releasing hormone (TRH), produced by the hypothalamus. The TSH acts upon the thyroid gland to stimulate the production to thyroxine (T4) and triiodothyronine (T3). The levels of T4 and T3 normally regulate the secretion of TSH by their negative feedback effect at the pituitary and possibly at the hypothalamus. Therefore, the measurement of circulation TSH levels is an important part of the investigation of disorders of the hypothalamic /pituitary/thyroid axis. The MYM TSH Quantitative employs microtiter ELISA with optimum sensitivity and specificity for the measurement of this hormone. SAMPLE COLLECTION AND HANDLING Well No. A1 B1 C1 D1 E1 F1 G1 H1 B1 7. 8. 9. Secure the desired number of coated wells in the holder. Dispense 50 uL of Standards, Controls or Serum samples. Dispense 100 uL of enzyme conjugate to each wells Incubate for 60 minutes at room temperature. Remove incubation mixture and rinse the wells 5 times with running tap water. Dispense 100 uL of Solution A and 100 uL of Solution B into each well. Incubate 30 minutes at room temperature. Stop reaction by adding 50 uL of 2 N HCl solution into each well. Read O.D. at 450 nm with a microwell reader. CALCULATION OF RESULTS 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). TSH 3.5 3 O.D. 450 nm 1. Biomedical Laboratories”, 1984. Never pipet by mouth. Avoid contact with skin. 2.5 2 1.5 1 0.5 0 0 10 20 30 40 50 TSH (uIU/mL) QUALITY CONTROL Each laboratory should utilize internal controls at several levels to monitor assay performance. The control should be treated as unknown. Results obtained should be in agreement with the assigned value of the control. The controls can be obtained from commercially available sources, but should not contain sodium azide as preservatives. LIMITATIONS 1. 2. For diagnostic purposes, the TSH values should be used as an adjunct to other data available to the physician. The MYM TSH Quantitative Kit has been designed so that the high dose hook effect will not interfere with TSH determination at levels up to 25 uIU/mL. Samples with TSH levels that fall above 25 uIU/mL standard should be diluted in order to obtain a more accurate value. EXPECTED VALUES 1. 2. It is recommended that each laboratory determine its own normal and abnormal range. As a guide from published literature, the value for TSH by immunoassay techniques generally place the adult euthyroid range below 6 uIU/mL. Extensive studies have been conducted on both normal and abnormal patient samples. The results of which are summarized below: Samples Euthyroid Hyperthyroid Hypothyroid 3. Hyperthyroid patients show no reaction to TRH. N 110 45 20 Range Upper Limit Less than Greater than uIU/mL 5.5 0.6 5.0 Some studies have indicated a circadian and episodic variation associated with TSH secretion. Newborn infants have substantially higher levels than are found later in life. INDICATION FOR QUANTITATIVE ASSAY OF TSH 1. 2. 3. 4. Normal Serum TSH levels of plasma TSH are generally reported as under 6 uIU per milliliter, with most assays being unable to detect levels less thatn 1 uIU per milliliter. Primary hypothyroidism in thyroprivic and goitrous varieties: An elevated TSH level is the most sensitive screen test for detecting hypothyroidism. The TSH level may be high before any clinical or laboratory test can show a low circulating level of T4. Hypothalmic-pituitary hypothyroidism: The TSH level is undetectable in the hypothalamic-pituitary hyprothyroidism minimal elevations of thyroid hormones in the blood prevent the TSH release following administration of TRH. In addition to possible evidence of intracranial disease hyposecretion of TSH is accompanied by hyposecretion of other pituitary hormones. A subnormal response of the serum TSH to the administration of TRH confirms the presence of hypothalamic-pituitary hypothyroidism. Thyroid status determination in person with low TSH level: The level of thyroid function can be determined by testing with thyrotropin releasing hormone (TRH). Hypothyroid patients react to TRH administration with greater release of TSH than do normal persons. PERFORMANCE CHARACTERISTICS Accuracy Recovery studies were performed by mixing an aliquot of pooled serum and TSH Standards. The TSH values were measured and percentage of recovery determined. Initial Values (uIU/mL) 2.5 2.5 2.5 25 Expected Values (uIU/mL) 6.25 13.75 26.25 34.16 Observed Values (uIU/mL) 6 14 28 36 Recovery (%) 96 102 106 105 Precision Inter-assay and intra-assay coefficient of variation were evaluated at three different pooled serum samples. N Mean (uIU/mL) S.D. (uIU/mL) C.V. (%) INTER-ASSAY Pool A Pool B 10 10 3.8 13.3 0.24 0.48 6.45 3.63 Pool C 10 45.5 2.01 4.43 N Mean (uIU/mL) S.D. (uIU/mL) C.V. (%) INTRA-ASSAY Pool A Pool B 10 10 3.59 12.47 0.28 0.32 7.71 2.54 Pool C 10 42.50 1.08 2.54 Specificity The following hormones were tested for crossreactivity of the assay: Hormone Tested HCG 400 mIU/mL LH 200 mIU/mL FSH 200 mIU/mL Produce Color Intensity Equivalent to TSH <0.20 uIU/mL <0.25 uIU/mL <.020 uIU/mL A linear study was performed to assess the sensitivity of the assay Initial Value (uIU/mL) 44 Dilution Folds 1:2 1:4 1:8 1:16 1:32 Expected Value (uIU/mL) 22.0 11.0 5.5 2.75 1.38 Observed Value (uIU/mL) 22.5 11.5 5.8 3.1 1.7 1:64 1:128 0.69 0.35 0.9 0.5 The sensitivity by this study was less than 1 uIU/mL. The minimal detectable concentration of TSH is estimated to be 0.5 uIU/mL. A minimal detectable concentration is defined as that concentration of TSH that corresponds to the absorbance that is two standard deviation from the mean absorbance of 20 serum samples with zero dose of TSH. REFERENCE 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 ThyroidStimulating Hormone: Neuro-regulation and Clinical Applications, Clin. Sci. Mol Med. 55 1 & 129, 1978 Feb 2005 (3)