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					                                                               concentration. These changes in the T3 level, however, are    1. Unopened test kits should be stored at 2-8ºC
                                                               not a true reflection of thyroid status.                         upon receipt and the microtiter plate should
                                                                                                                                be kept in a sealed bag with desiccants to
                                                               Principle of the test:                                           minimize exposure to damp air. The test kit
                                                               In the T3 EIA, a certain amount of anti-T3 antibody is           may be used throughout the expiration date
                                                               coated on microtiter wells. A measured amount of patient         of the kit (one year from the date of
                                                               serum, and a constant amount of T3conjugated to                  manufacturing). Refer to the package label
   TRIIODOTHYROXINE (T3)                                       horseradish peroxidase are added to the microtiter wells.        for the expiration date.
 ENZYME IMMUNOASSAY TEST                                       During incubation, T3 and conjugated T3 compete for the       2. Opened test kits will remain stable until the
                                                               limited binding sites on the anti-T3 antibody. After 60          expiration date shown, provided it is stored
            KIT                                                minutes incubation at room temperature, the wells are            as prescribed above.
                                                               washed 5 times by water to remove unbound T3                  3. A microtiter plate reader with a bandwidth of
Enzyme Immunoassay for the Quantitative Determination          conjugate. A solution of TMB is then added and incubated         10nm or less and an optical density range of
of Triiodothyroxine (T3) Concentration in Human Serum          for 20 minutes, resulting in the development of blue color.      0-2 OD or greater at 450nm wavelength is
                                                               The color development is stopped with the addition of 1N         acceptable     for    use    in    absorbance
Intended use:                                                  HCl, and the absorbance in measured spectro-                     measurement.
For the quantitative determination of total triiodothyroxine   photomertically at 450nm. The intensity of the color
(T3) in human serum                                            formed is proportional to the amount of enzyme present        Reagent preparation:
                                                               and is inversely related to the amount of unlabeled T3 in     1. All reagents should be brought to room
Introduction:                                                  the sample. By reference to a series of T3 standards             temperature (18-25ºC) before sue.
The Human thyroid gland is a major component of the            assayed in the same way, the concentration of T3 in the       2. To prepare T3-HRPO conjugate reagent, add
endocrine system. Thyroid hormones perform many                unknown sample is quantified.                                    0.1ml of T3-HRPO conjugate concentrate to
important functions. They exert powerful and essential                                                                          1.0ml of T4-HRPO conjugate diluent (1:10
regulatory influences on growth, differentiation, cellular     Materials and components:                                        dilution), and mix well. The amount of
metabolism, and general hormonal balance of the body, as       Materials provided with the test kits:                           conjugate reagent is stable at 4ºC at least for
well as on the maintenance of metabolic activity and the       • Goat Anti-Mouse IgG Coated Microtiter                          two weeks.
development of the skeletal and organ system.                    Well, 96 wells per pouch.
The hormones thyroxine (T4) and 3,5,3’ triiodothyroxine        • Reference standard set, ready to use.                       Assay procedure:
(T3) circulate in the bloodstream, mostly bound to the         • T3 Enzyme Conjugate Diluent, 13ml                           1. Secure the desired number of coated wells in
plasma protein, thyroxine binding globulin (TBG). The
                                                               • T3 Enzyme conjugate Concentrate (11X),                         the holder. Make data sheet with sample
concentration of T3 is much less than that of T4, but its                                                                       identification.
metabolic potency is much greater.                                                                                           2. Pipette 50µl of standard, sample, and
                                                               • Antibody Reagent, 7ml
T3 determination is an important factor in the diagnosis of                                                                     controls into appropriate wells.
thyroid disease. Its measurement has uncovered a variant       • TMB reagent , 11ml.
                                                                                                                             3. Dispense 50µl of the Antibody Reagent into
of hyper-thyrodism in thyrotoxic patients with elevated T3     • Stop Solution (1N HCl), 11ml.
                                                                                                                                each well, Mix thoroughly for 30 seconds.
values and normal T4 values. An increase in T3 without                                                                       4. Add 100µl of working conjugate reagent
an increase in T4 is frequently a forerunner of recurrent      Materials required but not provided:
                                                                                                                                into each well. Thoroughly mix for 30
thyrotoxosis in previously treated patients. The clinical      • Precision pipettes: 50µl~100µl and 1.0ml
                                                                                                                                seconds. It is very important to have
significance of T3 is also evident in patients in whom         • Disposable pipette tips.
                                                                                                                                complete mixing in this step.
euthyroidism is attributable only to normal T3, although       • Distilled water.                                            5. Incubate at room temperature (18-25ºC) for
their T4 values are subnormal.                                 • Vortex mixer or equivalent.                                    60 minutes.
T3 determination is also useful in monitoring both patients    • Absorbent paper or paper towel.                             6. Remove the incubation mixture by flicking
under treatment for hyper-thyrodism and patients who           • Graph paper.                                                   plate contents into a waste container.
have discontinued antithyroid drug therapy. It is especially   • Microtiter well reader.                                     7. Rinse and flick the microtiter wells 5 times
valuable in distinguishing between euthyroid and                                                                                with distilled water. (Please do not use tap
hyperthyroid subjects.                                         Specimen collection and preparation:                             water)
In addition to hyper-thyrodism, T3 levels are elevated in      Serum should be prepared from a whole blood specimen          8. Strike the wells sharply onto absorbent paper
women who are pregnant, and in women receiving oral            obtained by acceptable medical techniques. This kit is for       or paper towels to remove all residual water
contraceptives or estrogen treatment, paralleling TBG          use with serum samples without additives only.                   droplets.
increases in a manner analogues to T4 levels. Likewise, a                                                                    9. Dispense 100µl of TMB solution into each
reduction in TBG concentration decreases T3                    Storage of test kits and instrumentation:                        well. Gently mix for 10 seconds.
10. Incubate at room temperature in the dark for                                                                          5. Lieblicj J., Utiger R.D. J. Clin Invest. 1972;
    20 minutes without shaking.                                           3                                               51: 1939
11. Stop the reaction by adding 100µl of stop                            2.5
    solution (1N HCl) to each well.
12. Gently mix for 30 seconds. It is very                                 2                                               ATLAS Medical
    important to make sure that the blue color                                                                            Unit 4, William James House

    changes to yellow color completely.                                  1.5                                              Cowley Rd, Cambridge, CB4 0WX
13. Read optical density at 450nm with a                                                                                  Tel: ++44 (0) 1223 858 910
 microtiter well reader within 15 minutes.                                                                                Fax: ++44 (0) 1223 858 524
Important note:                                                                                                           PPI061A01
The wash procedure is critical. Insufficient washing will                                                                 Revision B (18.10.2004)
result in poor precision and falsely elevated absorbance                       0   2       4        6         8     10
                                                                                          Conc. (ng/ml)

Calculation of results:
1. Calculate the average absorbance values
   (A450) for each set of reference standards,
   controls and samples.
2. We recommend using proper software to                       Expected values and Sensitivity:
   calculate the results. If the software is not               Normal Range: 0.6~1.85ng/ml
   available, construct a standard curve by                    The minimal detectable concentration of T3 by this assay
   plotting the mean absorbance obtained for                   is estimated to be 0.2ng/ml
   each reference standard against its
   concentration in ng/ml on linear graph paper,               Limitations of the procedure
   with absorbance on the vertical (y) axis and                1. Reliable and reproducible results will be
   concentration on the horizontal (x) axis.                      obtained when the assay procedure is carried
3. Using the mean absorbance value for each                       out with a complete understanding of the
   sample, determine the corresponding                            package insert instructions and with
   concentration of T3 in ng/ml from the                          adherence to good laboratory practice.
   standard curve.                                             2. The wash procedure is critical. Insufficient
                                                                  washing will result in poor precision and
 Example of standard curve                                        falsely elevated absorbance reading.
 Results of typical standard run with optical density          3. Serum sample demonstration gross lipemia,
 reading at 450nm shown in the Y-axis against T3                  gross hemolysis, or turbidity should not be
 concentrations shown in the X-axis. This standard curve          used with this test.
 is for purpose of illustration only, and should not be used   4. The results obtained from the use of this kit
 to calculate unknowns. Each user should obtain his or            should be used only as an adjunct to other
 her own data and standard curve.                                 diagnostic procedures and information
                                                                  available to the physician.
         T3 (ng/ml)       Absorbance (450nm)

         0                2.685                                References:
         0.75             2.381                                1. Walker W.H.C. Introduction: An Approach
         1.5              2.028                                to Immunoassay. Clin. Chem. 1977;23:384
         3.0              1.502                                2. Kirkegaard C., Friis T. and Siersback-
         6.0              0.992                                Nielsen K. Acta Endocrinol. 1974; 77: 71
         10.0             0.518                                3. Wisdom G.B. Enzyme-Immunoassay. Clin
                                                               Chem. 1976; 22: 1243
                                                               4. Hoffenberg R. Medicine 1978; 8: 392

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