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Triiodothyronine (T3) Human ELISA Kit

show all "Triiodothyronine (T3) Human ELISA Kit "
  • Product Name
  • Triiodothyronine (T3) Human ELISA Kit

  • Cat.No.
  • DEIA1525
  • Pkg#Size
  • 96T
  • Intended use
  • For the quantitative measurement of Free Triiodothyronine (T3) concentrations in human serum.
  • General Description
  • L-Triiodothyronine, a thyroid hormone, circulates in blood almost completely bound (>99.5%) to carrier proteins. The main transport protein is thyroxine-binding globulin (TBG). However, only the free (unbound) portion of triiodothyronine is believed to be responsible for the biological action. Furthermore, the concentrations of the carrier proteins are altered in many conditions, such as pregnancy. In individuals with normal thyroid function, as the concentrations of the carrier proteins change, the total T3 levels change in concert so that the free thriiodothyronine (fT3) concentration remains constant. Thus, measurements of fT3 concentrations correlate more reliably with clinical status than total triiodothyronine levels.
  • Principle Of The Test
  • The Triiodothyronine (T3) Human ELISA Kit is based on the principle of a solid phase competitive enzyme-linked immunosorbent assay. Serum samples, standards, and T3-Enzyme Conjugate Working Reagent are added to wells coated with monoclonal T3 antibody. Free Triiodothyronine (T3) in the sample specimen and the T3 labeled conjugate compete for available binding sites on the antibody. After 60 minute incubation at room temperature, the wells are washed with water to remove unbound T3 conjugate. A solution of H2O2/TMB is then added and incubated for 20 minutes, resulting in the development of blue color. The intensity of the color formed is proportional to the amount of enzyme present and is inversely related to the amount of unlabeled Free Triiodothyronine (T3) in the sample. The color development is stopped with the addition of 3N HCl, and the absorbance is measured spectrophotometrically at 450 nm. By reference to a series of Free Triiodothyronine (T3) standards assayed in the same way, the concentration of Free Triiodothyronine(T3) in the unknown sample is quantified.
  • Reagents And Materials Provided
  • 1. T3 Antibody-Coated Microplate: 96 wells
    2. T3-Enzyme Conjugate: 10.5 ml, ready to use.
    3. T3 Reference Standards: 0, 0.9, 2.2, 5.0, 9.0, and 19.0 pg/ml. 1 set, 1.0 ml each, ready to use.
    For SI units: 1pg/ml x 1.536= pmol/l
    4. Color Reagent A: 13 ml
    5. Color Reagent B: 13 ml
    6. Stop solution (3N HCI): 10 ml
  • Materials Required But Not Supplied
  • 1. Pipette capable of delivering 50 μl volumes with a precision of better than 1.5%.
    2. Dispenser(s) for repetitive deliveries of 50 μl and 200 μl volumes with a precision of better than 1.5%.
    3. Microplate Reader with a bandwidth of 10 nm or less and an optical density range of 0-2 OD or greater at 450 nm wavelength is acceptable for use in absorbance measurement.
    4. Test tubes for dilution of enzyme conjugate and for mixing Color Reagent A with Color Reagent B.
    5. Absorbent paper of blotting the microplate wells.
    6. Timer.
    7. Quality control materials.
  • Storage
  • Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag with desiccants to minimize exposure to damp air. Opened test kits will remain stable until the expiration date shown, provided it is stored as described above.
  • Specimen Collection And Handling
  • Serum should be prepared from a whole blood specimen obtained by acceptable medical techniques. This kit is for use with serum sample without additives only. Serum samples may be refrigerated at 2-8°C for a maximum period of 48 hours. If the samples cannot be assayed within 48 hours, they may be stored at temperatures of –20°C for up to 30 days.
  • Reagent Preparation
  • Working Substrate Solution: Prepare immediately before use. To prepare H2O2/TMB solution, make a 1:1 mixing of Color Reagent A with Color reagent B up to 1 hour before use. Mix gently to ensure complete mixing. The prepared H2O2/TMB reagent should be made at least 15 minutes before use and is stable at room temperature in the dark for up to 3 hours. Discard excess after use.
  • Assay Steps
  • Before proceeding with the assay, bring all reagents, serum references and controls to room temperature (18-25°C).
    1. Format the microplates’ wells for each serum reference, control, and sample specimen to be assayed in duplicate.
    2. Pipette 50 μl of the appropriate serum reference, control, and specimen into the assigned well.
    3. Add 100 μl of T3-Enzyme Conjugate Solution to all wells.
    4. Swirl the microplate gently for 20-30 seconds to mix and cover.
    5. Incubate 60 minutes at room temperature.
    6. Remove the incubation mixture by emptying the plate content into a waste container. Rinse and empty the microtiter plate 5 times with distilled water. Strike the microtiter plate sharply onto absorbent paper or paper towels to remove all residual water droplets.
    7. Add 200 μl of Working Substrate Solution to all wells (see Reagents Preparation Section). Always add reagents in the same order to minimize reaction time differences between wells. Gently mix for 10 seconds.
    8. Incubate at room temperature in the dark for 20 minutes.
    9. Stop the reaction by adding 50 μl of 3N HCl to each well.
    10. Gently mix for 30 seconds. It is important to make sure that all the blue color changes to yellow color completely.
    11. Read absorbance at 450 nm with a microtiter well reader within 30 minutes.
  • Calculation
  • 1. Calculate the mean absorbance value (A450) for each set of reference standards, controls and samples.
    2. Construct a standard curve by plotting the mean absorbance obtained for each reference standard against its concentration in pg/ml on graph paper, with absorbance values on the vertical or Y axis, and concentrations on the horizontal or X axis.
    3. Use the mean absorbance values for each specimen to determine the corresponding concentration of free Triiodothyronine (T3) in pg/ml from the standard curve.

    Reference values:
    A study of euthyroid adult population was undertaken to determine expected values for the Triiodothyronine (T3) Human ELISA Kit. The mean (X) values, standard deviations (S.D.) and expected ranges (± 2 S.D.) are presented in the following table:
    It is important to keep in mind that establishment of a range of values which can be expected to be found by a given method for a population of “normal” persons is dependent upon several factors: the specificity of the method, the population tested, and the precision of the method in the hands of the analyst. For these reasons each laboratory should depend upon the range of expected values established by the Manufacturer only until an in-house range can be determined by the analysts using the method with a population indigenous to the area in which the laboratory is located.
  • Typical Standard Curve
  • The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.
  • Reference Values
  • A study of euthyroid adult population was undertaken to determine expected values for the Triiodothyronine (T3) Human ELISA Kit. The mean (X) values, standard deviations (S.D.) and expected ranges (± 2 S.D.) are presented in the following table.
    It is important to keep in mind that establishment of a range of values which can be expected to be found by a given method for a population of “normal” persons is dependent upon several factors: the specificity of the method, the population tested, and the precision of the method in the hands of the analyst. For these reasons each laboratory should depend upon the range of expected values established by the Manufacturer only until an in-house range can be determined by the analysts using the method with a population indigenous to the area in which the laboratory is located.
  • Sensitivity
  • The kit has a sensitivity of 0.05 pg/ml. The sensitivity was ascertained by determining the variability of the 0 pg/ml serum calibrator and using the 2 (95% certainty) statistic to calculate the minimum dose.
  • Specificity
  • The cross reactivity of the triiodothyronine antibody to selected substances was evaluated by adding the interfering substance to a serum matrix at various concentrations. The cross-reactivity was calculated by deriving a ratio between doses of interfering substance to dose of Triiodothyronine needed to displace the same amount of tracer.
  • Reproducibility
  • The within and between assay precision of ab108663 were determined by analyses on three different levels of pool control sera. The number, mean values, standard deviation and coefficient of variation for each of these control sera are shown in the following tables:
  • Limitations
  • 1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the package insert instructions and with adherence to good laboratory practice.
    2. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
    3. Serum samples demonstrating gross lipemia, gross hemolysis, or turbidity should not be used with this test.
    • Download Datasheet:
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