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Free T3 Kit ELISA

Détails pour le produit réf. ABIN996945, Fournisseur: Connectez-vous pour afficher
Kits with alternative reactivity to:
Gamme de detection
0-16 pg/mL
Seuil minimal de détection
0 pg/mL
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Specificité 97?%
Sensibilité 0.05 pg/mL
Matériel non inclus 1. Pipette capable of delivering 50 mL & 100 mL volumes with a precision of better than 1.5 %.
2. Dispenser(s) for repetitive deliveries of 1. 00 mL and 1. 50 mL volumes with a precision of better than 1. %.
3. Microplate washers or a squeeze bottle (optional).
4. Microplate Reader with 450 nm and 620 nm wavelength absorbance capability.
5. Absorbent Paper for blotting the microplate wells.
6. Plastic wrap or microplate cover for incubation steps.
7. Vacuum aspirator (optional) for wash steps.
8. Timer.
9. Quality control materials
Autre désignation Free-T3
Sujet 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. Further, the concentrations of the carrier proteins are altered in many clinical conditions, such as pregnancy. In normal thyroid function as the concentrations of the carrier proteins alters, the total triiodothyronine level changes so that the free triiodothyronine concentration remains constant. Thus, measurements of free triiodothyronine concentrations correlate more reliably with clinical status than total triiodothyronine levels. For example, the increase in total triiodothyronine levels associated with pregnancy, oral contraceptives and estrogen therapy result in higher total T3 levels while the free T3 concentration remains basically unchanged.

This microplate enzyme immunoassay methodology provides the technician with optimum sensitivity while requiring few technical manipulations in a direct determination of free T3. In this method, serum reference, patient specimen, or control is first added to a microplate well. Enzyme-T3 conjugate (analog method) is added, then the reactants are mixed. A competition reaction results between the enzyme conjugate and the free triiodothyronine for a limited number of antibody combining sites immobilized on the well. After the completion of the required incubation period, the antibody bound enzyme- triiodothyronine conjugate is separated from the unbound enzyme-triiodothyronine 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 color. The employment of several serum references of known free triiodothyronine concentration permits construction of a graph of activity and concentration. From comparison to the dose response curve, an unknown specimen's activity can be correlated with free triiodothyronine concentration.

Quality Control:
Each laboratory should assay controls at levels in the hypothyroid, euthyroid and hyperthyroid range for monitoring assay performance. These controls should be treated as unknowns and values determined in every test procedure performed. Quality control charts should be maintained - follow the performance of the supplied reagents. Pertinent statistical methods should be employed - ascertain trends. Significant deviation from established performance can indicate unnoticed change in experimental conditions or degradation of kit reagents. Fresh reagents should be used - determine the reason for the variations.

Note 1:
Do not use reagents beyond the kit expiration date.
Note 2:
Opened reagents are stable for sixty (60) days when stored at 2-8 °C.

Volume d'échantillon 50 μL
Durée du test 1 - 2 h
Préparation des réactifs
  1. Wash Buffer Dilute contents of Wash Concentrate to 1000 mL with distilled or deionized water in a suitable storage container. Store at room temperature 20-27 °C for up to 60 days.
    2. Working Substrate Solution Pour the contents of the amber vial labeled Solution A into the clear vial labeled Solution B. Place the yellow cap on the clear vial for easy identification. Mix and Label accordingly. Store at 2 -8 °C
    Note: Do not use the working substrate if it looks blue.
Préparation de l'échantillon

The specimens shall be blood serum in type and the usual precautions in the Collection of venipuncture samples should be observed. The blood should be collected in a plain red-top venipuncture tube without additives or gel barrier. Allow blood to clot. Centrifuge the specimen to separate the serum from the cells. Specimen may be refrigerated at 2-8 °C for a maximum period of 48 hours. If the specimen(s) cannot be assayed within 48 hours, the sample(s) may be stored at temperatures of -20 °C for up to 30 days. When assayed in duplicate, 0.100 mL of the specimen is required.

Procédure de l'essai

Before proceeding with the assay, bring all reagents, serum, references and Controls to room temperature 20 -25 °C

In order for the assay results to be considered valid the following criteria should be met:
1. The absorbance OD of calibrator A should be >1.3
2. Four out of six quality control pools should be within the established ranges.

1. It is important that the time of reaction in each well is held constant for reproducible results.
2. Pipetting of samples should not extend beyond ten (10) minutes to avoid assay drift.
3. Highly lipemic, hemolyzed or grossly contaminated specimen(s) should not be used.
4. If more than one (1) plate is used, it is recommended to repeat the dose response curve.
5. The additional of substrate solution initiates a kinetic reaction, which terminated by the addition of the stop solution. Therefore, the substrate and stop solution should be added in the same sequence to eliminate any time-deviation during reaction.
6. Plate readers measure vertically. Do not touch the bottom of the wells.
7. Failure to remove adhering solution adequately in the aspiration or decantation wash steps(s) may result in poor replication and spurious results.
8. Use components from the same lot. No intermixing of reagents from different batches.
9. Accurate and precise pipetting, as well as following the exact time and temperature requirements prescribed are essential. Any deviation from DAI IFU may yield inaccurate results.
10. All applicable national standards, regulations and laws, including, but not limited to, good laboratory procedures, must be strictly followed to ensure compliance and proper device usage.
11. It is important to calibrate all the equipment e.g. Pipettes, Readers, Washers and/or the automated instruments used with this device.

Calcul des résultats

A dose response curve is used to ascertain the concentration of free triiodothyronine in unknown specimens.
1. Record the absorbance obtained from the printout of the microplate reader as outlined in Example

2. Plot the absorbance for each duplicate serum reference versus the corresponding fT3 concentrationin pg/mL on linear graph paper (do not average the duplicates of the serum references before plotting).

3. Draw the best fit curve through the plotted points.
4. To determine the concentration of FT3 for an unknown, locate the average absorbance of the duplicates for each unknown on the vertical axis of the graph, find the intersecting point on the curve and read the concentration in pg/mL from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated) In the following example, the average absorbance (
1.855) (intersects the standard curve at (
2.1pg/mL) FT3 concentration (See Figure 1).
cc A. Precision The within and between assay precisions of the FT3 DAI ELISA test system were determined by analyses on three different levels of pool control sera. The number (N) mean values (X), standard deviation (sigma) and coefficient of variation (C.V.) for each of these control sera are presented in Table 2 and Table

3. TABLE 2 Within Assay Precision (Values in pg/mL) Sample N X sigma C.V. Low 24
1.85 0.09
4.9 % Medium 24
4.49 0.16
3.6 % High 24
8.00 0.25
3.1 % TABLE 3 Inter Assay Precision (in pg/mL) Sample N X sigma C.V. Low 12
2.16 0.29 1
3.1 % Medium 12
5.09 0.40
7.9 % High 12
9.13 0.94
1.2 % *As measured in twelve experiments in duplicate. B. Accuracy The FT3 DAI ELISA test system was compared with a coated tube radioimmunoassay analog method. Biological specimens from hypothyroid, euthyroid and hyperthyroid populations were used (The values ranged from 0.1pg/mL - 14pg. mL) . The total number of such specimens was 15
1. The least square regression equation and the correlation coefficient were computed for this FT3 DAI ELISA method in comparison with the reference method. The data obtained is displayed in Table
4. TABLE4 Least Square Regression Method Correlation Mean (X) Regression Analysis Correlation Coefficient This Method (Y)
3.05 y=0.35+0.922(x) 0.902 Reference (X)
2.92 dose of interfering substance to dose of triiodothyronine needed to displace the same amount of conjugate. Substance Cross Reactivity Concentration I-triidothyronine
1.0000 - I-Thyroxine <0.0002 10μ^ Iodothyrosine <0.0001 10μ^ Diidothyrosine <0.0001 10μ^ Diiodothyronine <0.0001 10μ^ Phenylbutazone <0.0001 10μ^ Sodium Salicylate <0.0001 10μL
A study of euthyroid adult population was undertaken to determine expected values for the FT3 DAI ELISA test system. The mean values (X), standards deviations (sigma) and expected ranges (±2sigma.) are presented in Table 1 TABLE I Expected Values for the Free T3 ELISA Test System (in pg/mL) Adult (110 specimens) Pregnancy (75 specimens) Mean X
3.0 Standard Deviation (sigma) 0.7 0.6 Expected Ranges (±2sigma)
1.4 -
1.8 -
4.2 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 a multiplicity of factors: the specificity of the method, the population tested, and the precision of the method in the hands of the analysts. 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.

1. Laboratory results alone are only one aspect for determining patient care and should not be the sole basis for therapy, particularly if the results conflict with other determinants.

2. For valid test results, adequate controls and other parameters must be within the listed ranges and assay requirements.

3. If test kits are altered, such as by mixing parts of different kits, which could produce false test results, or if results are incorrectly interpreted, DAI shall have not liability.
4. If computer controlled data reduction is used to calculate the results of the test. It is imperative that the predicted values for the calibrators fall within10 % of the assigned concentrations.
5. If a patient for some reason reads higher that the highest calibrator report as such (e.g >16 pg/mL). Do not try to dilute the sample. TBG variations in different matrices will not allow FT3 hormone to dilute serially.
6. Several drugs are known to affect the binding of Triiodothyronine to the Thyroid hormone carrier proteins or metabolism to T3 and complicate the interpretation of FT3 results (3).
7. Circulating autoantibodies to T3 and hormone binding inhibitors may interfere (4).
8. Heparin has been reported to have in vivo and in vitro effects on FT3 concentration (5). Therefore, do not obtain samples in which this anticoagulant has been used.
9. In severe nonthyroidal illness (NTI) the assessment of thyroid status becomes very difficult. TSH measurements are recommended to identify thyroid dysfunction (6).
10. Familiar dysalbuminemic conditions may yield erroneous results on direct free T3 assays (7). "NOT INTENDED FOR NEWBORNS SCREENING"

Restrictions For Research Use only
Stock 4 °C
Date de péremption 12-14 months
Reference: Background Lundberg, Jagenburg, Lindstedt et al.: "Heparin in vivo effect on free thyroxin." dans: Clinical chemistry, Vol. 28, Issue 5, pp. 1241-2, 1982 (PubMed).

Lalloz, Byfield, Himsworth: "Hyperthyroxinaemia: abnormal binding of T4 by an inherited albumin variant." dans: Clinical endocrinology, Vol. 18, Issue 1, pp. 11-24, 1983 (PubMed).

Bhagat, Garcia-Webb, Watson et al.: "Interference in radioimmunoassay of total serum thyroxin and free thyroxin due to thyroxin-binding autoantibodies." dans: Clinical chemistry, Vol. 29, Issue 6, pp. 1324-5, 1983 (PubMed).

Melmed, Geola, Reed et al.: "A comparison of methods for assessing thyroid function in nonthyroidal illness." dans: The Journal of clinical endocrinology and metabolism, Vol. 54, Issue 2, pp. 300-6, 1982 (PubMed).

Wenzel: "Pharmacological interference with in vitro tests of thyroid function." dans: Metabolism: clinical and experimental, Vol. 30, Issue 7, pp. 717-32, 1981 (PubMed).

Pedersen: "A systematic study of variables affecting protein binding of thyroxine and triiodothyronine in serum." dans: Scandinavian journal of clinical and laboratory investigation, Vol. 34, Issue 3, pp. 247-55, 1975 (PubMed).