|
|
      |
|
|
|
| GENTAUR • Belgium : + 32 2 7325688 • France : 01 43250150 • Italy : 02 36006593 • Germany : +49 241 6085 13140 | |
| |||||
|
|
EK-34A051101
FT4 ELISA KIT (REF: EK-34) 475 Euro The FT 4 ELISA system provides quantitative in vitro determination of free thyroxine (FT4) in human serum. FT4 can be assayed in the range of 0-100 pmol/l (0-7.77 ng/ml) using 25 µl serum samples.Introduction Circulating thyroid hormones (thyroxin, T 4 and triiodothyronine, T3) are distributed into two, a major, protein-bound, and a minor, (0.03 % of T4 and 0.3 % of T3) free fractions. Variations in total thyroid hormone in blood may result from either changes of binding proteins concentrations, or thyroid hormone production. Thyroid disorders are existing only if a net change of free unbound fractions occur persistently. Therefore the clinical utility of total T4 and T3 is dependant on the knowledge of functional levels of binding proteins.Serum level of free T 4 (FT4) correlates very well with secretion and metabolism rate of T4, and has been recommended as the most reliable and meaningful diagnostic indicator of thyroid diseases, mostly in conflicting or borderline instances. Apparent FT4 levels, however, are very sensitive to the analytical method due to the sophisticated multiple equilibrium between various protein compartments of T4.Principle of method This assay is based on the competition between FT 4 present in standards and samples - and conjugate (enzyme-T4) for a limited number of binding sites on anti-thyroxin antibodies. After the completion of the required incubation period, the antibody bound enzyme-conjugate is separated from the unbound enzyme-conjugate by aspiration or decantation. The activity of the enzyme present on the surface of the well is quantitated by reaction with a suitable substrate to produce colour.After the addition of a ready-to-use tetramethyl-benzidine (TMB) / peroxide substrate the signal is measured in an ELISA photometer at 450 nm wavelength. The concentration of antigen is directly proportional to the optical density measured in the wells. The unknown concentration of FT 4 in patient samples is read of a calibration curve constructed by plotting binding values against a series of calibrators containing known amounts of FT4.Contents of the kit 1 . 1 bottle CONJUGATE (5.5 ml), ready to use, containing a solution of thyroxine conjugated to horseradish peroxidase in buffer.Do not expose to direct sunlight! Store unopened at 2-8 °C until expiration date. Store opened bottle at 2-8 °C for up to 60 days. 2. 6 vials STANDARD (6 x 0.5 ml), ready to use, containing 0 (S1), 6 (S2), 12 (S3), 25 (S4), 50 (S5) 100 (S6) pmol/l in human serum with 0.1 % NaN3.(1 pmol/l = 0.0777 ng/dl) Store unopened at 2-8 °C until kit expiration date. Store opened vials at 2-8 °C for up to 60 days. 3. 1 vial CONTROL SERUM (0.5 ml), lyophilised human serum with 0.1% NaN 3.The concentration of the control serum is specified in the quality certificate enclosed. Store unopened at 2-8 °C until kit expiration date. See Preparation of reagents.4. 1 bottle SUBSTRATE (25 ml) ready to use, in brown plastic bottle. Do not expose to direct light! Store unopened at 2-8 °C until expiration date. Store opened bottle at 2-8 °C for up to 60 days. 5. 1 piece MICROTITER PLATE, ready to use. 12 strips, packed in an air-tight foil.Store at 2-8 °C until expiration date. 6. 1 bottle WASH BUFFER CONCENTRATE (20 ml), 0.01 % merthiolate.Store at 2-8 °C until expiration date. See Preparation of reagents.7. 1 bottle STOP REAGENT (6 ml) ready ro use, 1M sulfuric acid Store at 2-8 °C until expiration date. Plate map Quality certificate Pack leaflet Plastic foil Materials, tools and equipment required Precision pipettes with disposable tips (25, 50, 100, 200 and 300 µl), distilled water, vortex mixer, shaker, plastic tray, absorbent tissue, ELISA photometer Recommended tools and equipment Repeating pipettes, multi-channel ELISA pipettes Specimen collection and storage Serum samples can be prepared according to common procedures used routinely in clinical laboratory practice. Samples can be stored at 2-8 °C if the assay is carried out within 24 hours, otherwise aliquots should be prepared and stored deep frozen (-20°C). Frozen samples should be thawed and thoroughly mixed before assaying. Repeated freezing and thawing should be avoided. Do not use lipemic, hemolyzed or turbid specimens. Preparation of reagents, storage Add the wash buffer concentrate (20 ml) to 600 ml distilled water to obtain 620 ml wash solution. Upon dilution store at 2-8°C for up to 60 days. Add 500 µl distilled water to the lyophilised control serum. Mix gently with shaking or vortexing (foaming should be avoided). Ensure that complete dissolution is achieved, and allow the solution to equilibrate at room temperature for at least 20 minutes. Store at 2-8°C for up to 60 days. The actual expiry date of the kit is given on the package label and in the quality certificate. CAUTION! Equilibrate all reagents and serum samples to room temperature. Mix all reagents and samples thoroughly before use. Avoid excessive foaming. Assay procedure (For a quick guide , refer to Table 1.) Table 1. Assay Protocol, Pipetting Guide (all volumes in microlitres)
Calculate the average OD for each pair of duplicates. Draw the standard curve on a lin-lin graph paper by plotting calculated OD of each standard level (ordinate) against the respective concentration (abscissa). Obtain sample values by interpolation of sample OD on the standard curve. Specific binding values can be calculated for each standard and sample according to the following equation: 100)()(,,,%/ 1620×-=ODSODSCSSBBxTable 2. Typical assay data
00,511,522,50102030405060708090100 Figure 1: A typical standard curve (Do not use to calculate unknown samples!)Characterization of assay Typical assay parameters Maximum Absorbance (S1 OD) > 1.2 Sensitivity For the analytical sensitivity 2.15 pmol/l has been obtained by assaying 15 replicates of the zero standard. The sensitivity has been determined as the concentration corresponding to the sum of the mean OD and its double standard deviation. As Specificity T3 and rT3 were added in 5 different concentrations to T4 free standard (S1=B0) and the concentration of FT4 was measured. The cross reactivity are shown below.
DL – detection limit Precision 7 patient samples were assayed in 15 replicates to determine intra-assay precision. Values obtained are shown below.
Reproducibility To determine inter-assay precision 7 samples were measured in duplicates in 10 independent assays by 3 operators using different kit batches. Values obtained are shown below.
Recovery Recovery was defined as the measured increase expressed as per cent of expected increase upon spiking serum samples with known amount of T4. 85.3 ± 5.9 % (mean ± SD) was obtained for 5 samples. Expected Values It is recommended that each laboratory establish its own reference intervals. The expected values presented here are based on testing of apparently healthy blood donors. Samples were measured in duplicates using different kit lots. In a population (n=199) of adult female and male blood donors (ages: mean 38.09 ± 10.02, range 17 - 61) serum concentrations of FT4 were 15.6 ± 2.07 pmol/l (mean ± SD).a guide (mean ± 2*SD), 11.4 – 19.7 pmol/l reference range was obtained from normal patients based on statistical consideration only. Taking into consideration not only statistical results but clinical practice as well, a more realistic reference range can be recommended 10-22 pmol/l.Procedural notes A thorough understanding of this package insert is necessary for successful use of the kit. Reliable results will only be obtained by using precise laboratory techniques and accurately following the package insert. Use a clean disposable pipette tip for each reagent, Standards, Control or specimen. Pipetting of samples should not extend beyond 10 minutes to avoid assay drift. Always add reagents in the same order to minimize reaction time differences between wells. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. Avoid microbial contamination of reagents, especially of the Conjugate solution and TMB substrate: -Do not leave the cap off of the storage bottle for prolonged periods of time. -Never pipette directly from the bottle. Always pour just the necessary volume into a separate container for use then discard the excess after use. The results should be read within 30 minutes of adding the Stop solution. Additional information Components from various lots or from kits of different manufacturers should not be mixed or interchanged. Biohazard Human blood products used in the kit have been obtained from healthy human donors. They were tested individually by using approved methods (EIA, enzyme immunoassay), and were found to be negative, for the presence of both Human Immunodeficiency Virus antibody (Anti-HIV-1) and Hepatitis B surface Antigen (HBsAg). Care should always be taken when handling human specimens to be tested with diagnostic kits. Even if the subject has been tested, no method can offer complete assurance that Hepatitis B Virus, Human Immunodeficiency Virus (HIV-1), or other infectious agents are absent. Human blood samples should therefore be handled as potentially infectious materials.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
© 2005 GENTAUR bvba |
