THYROID UPTAKE ENZYME IMMUNOASSAY KIT; Page 1
Atlas Link
12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
THYROID UPTAKE ENZYME IMMUNOASSAY KIT
CATALOG NUMBER 3177
INTENDED USE
The Atlas Link Thyroid Uptake is used for the assesment of unsaturated Thyroxine Binding Capacity in human serum. The Atlas Link Thyroid Uptake test is used in conjunction with the Atlas Link T4 Enzyme Immunoassay to determine the Free Thyroxine Index in human serum.
EXPLANATION OF THE TEST
The thyroid gland secretes two hormones, thyroxine (T4) and triiodothyronine (T3). Thyroxine is the most commonly measured hormone for the diagnosis of thyroid function. Thyroxine has its primary influence in protein synthesis and oxygen consumption in virtually all tissues but it is al important for growth, development and sexual maturation(1).
Primary hypothyroidism results in underproduction of thyroid hormones by the thyroid gland and consequently and abnormally low circulating T4 and T3 concentration in the blood. Primary hyperthyroidism leads to excessive production of thyroid hormones and resulting elevation of T4 and T3 concentrations.
Thyroid hormones are secreted into the bloodstream where they become bound to serum proteins for transport to the cells. The major transport protein is Thyroxine Binding Globulin (TBG) which normal accounts for 80% of the bound hormone. Other thyroid hormone binding proteins are Thyroxine Binding Prealbumin and Albumin(2,3). Most of the serum thyroid hormones are bound to these transport proteins, leaving only about 0.03% of the T4 and 0.3% of the T3 free to exert their effect on cells. It is this very low concentration of free hormone which is the metabolically active fraction and the best indicator of patient thyroid status. Simply determining the total T4 concentration fails to take into account variations in TBG levels can vary which can affect the free T4 concentration. TBG levels can vary for reasons incidental to the patient’s thyroid status such as the presence of certain drugs and steroid hormones, pregnancy, and various non-thyroidal Uptake test was developed to produce and indirect measure of the unsaturated TBG in the specimen. The product of the total T4 concentration and the Thyroid Uptake value is called a Free Thyroxine index (FTI)(4). The FTI correlates more closely with Free T4 concentration, and is better method of monitoring thyroid function and diagnosing thyroid illness than a Total T4 determination alone.
The Atlas Link Thyroid Uptake test is a rapid sensitive method for measuring the unsaturated binding capacity of serum binding proteins. The long shelf-life of this product together with the elimination of radioisotopes, radiation counters, and necessary licensing requirements make this method applicable to all potential users both large and small laboratories.
PRINCIPLE OF THE METHOD
The Atlas Link Thyroid Uptake test is a solid phase competitive enzyme immunoassay. The method utilizes a highly specific anti-T4 antibody bound to a solid support and a T4-labeled enzyme conjugate. Patient serum , Euthyroid Serum Calibrator, and controls are pipetted into antibody coated wells, A fixed quantity of conjugate reagent containing exogenous T4 and enzyme -labeled T4 is then added to the wells. The exogenous T4 from the conjugate reagent is able to bind to both the antibody on the well and available sites on serum thyroxine-binding proteins. The enzyme labeled conjugate is capable to binding only to the antibody. In a hyperthyroid specimen, the endogenous level of T4 is high; therefore, there are fewer serum protein binding sites available for binding the exogenous T4. The opposite is true for a hypothyroid specimen. An equilibrium is established between the T4 bound to antibody, the T4 bound to serum binding proteins, and the enzyme labeled T4 bound to the antibody. After a brief incubation, the wells are decanted and washed. Substrate for the enzyme is added. The product of the enzyme-substrate reaction forms a colored complex when the stopping reagent is added. The absorbance is measured spectrophotometrically at 492nm. The Thyroid Uptake of the patient serum sample is calculated as [(Abs(eu)/Abs(x)TU(Ref)], where Abs(EU) is the absorbance for the Euthyroid Calibrator; Abs(x) is the absorbance for the patient or control serum and TU(Ref) is assigned % Uptake for the Euthyroid Calibrator.
REAGENTS
Note: Store all reagent at 2-8 C.
1. EUTHYROID SERUM CALIBRATOR, (2mL). Each vial contains human serum, thyroxine and .1% sodium azide as preservatives.
2. HYPOTHYROID SERUM CONTROL, (2mL). Each vial contains human serum, thyroxine and .1% sodium azide as preservatives.
3. HYPERTHYROID SERUM CONTROL, (2mL). Each vial contains human serum, thyroxine and .1% sodium azide as preservatives.
CAUTION: HANDLE AS IF CAPABLE OF TRANSMITTING HEPATITIS AND HUMAN IMMUNODEFICIENCY VIRUS. Source materials from which the calibrator and controls were derived were found non-reactive for HBsAg and HIV when tested with licensed reagents. However, no known test can assure that a product derived from a human source does not contain these viruses.
4. ANTIBODY-COATED WELLS, (96 wells). Each package contains mouse monoclonal antibody-coated polystyrene wells.
5. TU ENZYME CONJUGATE, (20mL or 60mL). Each vial contains thyroxine-labeled alkaline phosphatase (source: calf intestine), Tris-buffered saline, bovine serum albumin, and .1% sodium azide as preservative.
6. SUBSTRATE REAGENT, (20mL or 100mL). Each bottle contains carbonate buffer, magnesium chloride, .1% sodium azide, aminoantipyrene and phenyl phosphate.
7. STOPPING REAGENT, (20 mL or 150 mL). Each bottle contains phosphate buffer, .1% sodium azide, and potassium ferricyanide.
8. WASH BUFFER CONCENTRATE, (55 mL or 200 mL). Each bottle contains Tris-buffered saline, bovine serum albumin and .1% sodium azide.
WARNING: Care should be taken in discarding the reagents containing Sodium Azide which may react with lead and copper plumbing from explosive metal azides. On disposal, flush with a large volume of water to prevent azide build-up.
SPECIMEN COLLECTION AND HANDLING
1. Handle all blood and serum as if capable of transmitting Hepatitis and Human Immunodeficiency Virus.
2. Fresh serum is required for use in the assay procedure.
3. Serum specimens may be stored tightly capped and refrigerated (2-8C) for 7 days. If storage time exceeds seven days then frozen storage (-20C) for up to 30 days is recommended.
4. AVOID MULTIPLE FREEZE THAW CYCLES FOR ANY SPECIMEN.
5. Prior to assay, frozen sera should be completely thawed and mixed well.
6. Do Not Use Grossly Lipemic Specimens.
7. Moderately lipemic, hemolyzed and icteric specimens will not interfere with the assay.
MATERIALS REQUIRED BUT NOT PROVIDED
1. Calibrated micropipettes (25,100 microliters)
2. Absorbent paper.
3. Microwell strip or plate reading spectrophotometer, capable of reading at wavelength of 492 nm.
4. 100 or 500 mL graduated cylinder.
5. Beaker or flask for dilution of the Wash Buffer Concentrate.
REAGENT PREPARATION
- Wash Buffer Concentrate. Dilute the Wash Buffer Concentrate 1:10 with distilled water. To dilute the entire bottle, add the contents of the bottle to either 495 mL or 1800 mL of distilled water.
- All other reagents are supplied ready to use.
- Prior to use, warm all reagents to ambient temperature by allowing them to stand 1 hour on the bench top, or by briefly warming them at 37C in water bath. Gently mix all reagents.
- Do not use reagents beyond their expiration date.
- Do not mix or interchange reagent lots with any other kit lots.
PROCEDURAL NOTES
1. It is recommended that standards, controls and samples be run in duplicate for optimal performance.
2. When pippetting reagents, maintain a consistent order of addition from well to well. This will ensure equal incubation times for all wells. All reagents should be kept covered when not in use.
3. Read the absorbances within one hour after completing the assay.
4. Controls should be run with every assay.
5. If the microwell reading spectrophotometer requires a reagent blank, mix 100 l of Substrate Reagent with 100 l of Stopping Reagent in an unused well.
6. All residual wash buffer must be drained from the wells by efficient aspiration or blotting by tapping the plate forcefully on absorbant paper before proceeding to the next step. Never insert absorbant paper directly into the wells.
7. Take special care that the strips do not fall out of the holder when decanting.
8. The user of this kit should have read and understood the package insert prior to running the test. Strict adherence to the protocol is necessary to obtain reliable results with this as with any other immunoassay. In particular, careful reagent handling and storage, pipetting, and decanting are essential for achieving accurate and precise Thyroid Uptake determinations.
9. The Euthyroid Calibrator must be run with each assay series. Improper handling of patient samples may cause spurious results. Always mix the thawed samples thoroughly prior to assay. Avoid using old or mistreated serum specimens. Sample degradation as well as multiple freeze-thaw cycles may cause inaccurate Thyroid Uptake determinations. Patient specimens should be assayed as soon as possible after obtaining sample.
10. All incubation must be at room temperature. Do not shake the plate.
ASSAY PROCEDURE
Review Procedural Notes Before Running Assay
1. Pipette 25 l of serum sample, calibrator and controls into the appropriate wells.
2. Pipette 100 l of the TU Enzyme Conjugate into each well.
3. Incubate at room temperature for 30 minutes.
4. Decant or aspirate to discard contents of wells.
5. Fill the wells with the Wash Buffer previously prepared (See Reagent Preparation) and decant or aspirate completely. Wash the wells in this fashion three more times. Decant and thoroughly drain on absorbant paper or aspirate thoroughly to remove all liquid from wells.
6. Pipette 100 l of Substrate Reagent into each well.
7. Incubate at room temperature for 20 minutes.
8. Pipette 100 l of the Stopping Reagent into each well
9. Read the absorbance values at 492 nm. All wells must be read within one hour or less. (See procedural note #5 for Blanking instructions) (If available follow application method for specific automated analyzer).
RESULTS
Percent Uptake Calculation
1. The percent uptake for each control or patient sample is calculated from the following equation:
% Uptake = Abs (EU)TU (REF)
Abs (x)
where:Abs (EU) = Absorbance for Euthyroid Calibrator
Abs (x) = Absorbance of the control or patient sample
TU (REF) = percent uptake of Euthyroid Calibrator
(see vial label)
CAUTION: Control values should not deviate from established range for the assay to be valid. (See vial label for ranges)
Calculation of Free Thyroxine Index
1. Determine the serum Total T4 concentration in ug% for each sample using the Atlas Link T4 kit.
2. Determine the percent uptake using the equation above.
3. The Free Thyroxine index (FTI) can then be calculated as follows:
FTI = Total T4 (ug%) x (%Uptake)
100
REPRESENTATIVE DATA TABLE
Sample Absorbance %Uptake
Euthyroid Calibrator.0.970
(TU Ref=38%)1.018
Hypothyroid Control1.310
1.273 29%
Hyperthyroid Control0.713
0.706 53%
Patient 1 1.345
1.263 29%
Patient 21.075
1.024 36%
Patient 30.759
0.733 51%
LIMITATION OF THE PROCEDURE
Thyroid Uptake is used, in conjunction with Total T4, to determine the Free Thyroxine Index which is a good indicator of thyroid function. Thyroid Uptake alone is not an accurate reflection of clinical thyroid status.
The Atlas Link Thyroid Uptake kit must be run with Atlas Link T4 kit for accurate FTI determinations. The use of other Total T4 assay procedures may result in erroneous values.
It has been determined that FTI levels correlate well with the thyroid status of the patient. However, there are situations that can make the interpretations of FTI results complex and FTI measurements should not be the sole basis for determining thyroid status.
Normal thyroid hormone levels do not include thyroid disease, and diffuse or nodular thyroid enlargements may be seen in euthyroid patients(5).
TBG concentrations have been reportedly altered by increased estrogens, anabolic steroids, androgens, glucocorticoids and pregnancy(6,7,8). Major illness, surgical stress, genetic deficiency and hepatitis can also affect TBG concentrations, with possible consequences on FTI levels.
Familial dysalbuminemic hyperthyroxinemia(9), auto-antibodies to T4(10) and analbuminemia(11) can result in elevation of Total T4 but should not affect FTI levels unless the patients are receiving T4 treatment(11).
CLINICAL SIGNIFICANCE
Thyroid Uptake is not a reliable single test of thyroid function since it is influenced by both the concentration and saturation of thyroxine binding proteins. Alterations in the concentration of serum binding proteins will generally result in a corresponding change in Total T4 concentration while the physiologically active Free T4 level remain largely unchanged in an euthyroid individual. Rather than relying on the Thyroid Uptake result alone, a more accurate assessment of thyroid status can be made by determining the Free T4 concentration or by calculation of the Free Thyroxine index (FTI). The FTI is the product of the Total T4 and Thyroid Uptake values. Elevated FTI concentration are indicated of Hyperthyroidism and low levels are indicative of Hypothyroidism.
The Atlas Link Thyroid Uptake test will give results similar to T3 Uptake tests. In certain cases, the Atlas Link Thyroid Uptake test may better diagnose thyroid status. For example, in the disorder known as Familial Dysalbuminemic Hyperthyroxemia, the use of T3 Uptake would give a false dianosis of thyrotoxicosis but the Atlas Link Thyroid Uptake would not(12).
LABORATORY QUALITY CONTROL
a. Do not mix or interchange reagent lots with any other kit or different kit lots.
b. Do not use reagents beyond the expiration date jprinted on each vial or bottle.
c. Each laboratory should establish its own criteria for precision and accuracy by running controls in the hypothyroid and hyperthyroid ranges.
d. Trend charts and statistical methods should be updated to assure that performance is reliable and consistent from lot to lot.
e. If the hypothyroid or hyperthyroid controls are not in their established range, the test results may not be valid.
EXPECTED VALUES
A. It is recommended that each laboratory establish its own normal range based on a representative sampling of the local population. The following Thyroid Uptake (% Uptake) values were determined and may be used as initial guideline ranges:
Euthyroid 33-43 % Uptake
Hyperthyroid > 43 % Uptake
Hypothyroid < 33 % Uptake
B. A normal FTI range of 2.0 - 4.1 was established by using the Atlas Link T4 and Atlas Link Thyroid Uptake enzyme immunoassay kits. Each laboratory should establish its own normal range.
PERFORMANCE CHARACTERISTICS
1. Correlation with other methods
122 patient samples were assayed using both the Corning Magic and Atlas Link Thyroid Uptake Kits. The assays diagnosed the patients as follows:
Method Hypothyroid Euthyroid Hyperthyroid
Corning Magic 30% 58% 12%
Diagnostic 30% 57% 13%
Automation
The FTI values of 122 serum samples were calculated using the Atlas Link T4 and Thyroid Uptake Kits, and commercially available Total T4 and Thyroid Uptake Kits. The Correlation is as follows:
Correlation
Method Coefficient Slope Intercept
RA/RIA 0.970.96 - 0.03
2. Precision
a. Intra-assay Precision was determined by assaying 14 replicated of each of the three serum pools.
PoolNo of Mean Standard Coefficient of
Replicates (ng/dl) Deviation Variation(%)
A 14 27.6%0.842 3.1
B14 33.4%0.928 2.8
C14 40.5%1.091 2.7
b. Inter-assay Precision was determined by assaying duplicates of three serum pools in ten separate runs.
PoolNo of Mean Standard Coefficient of
Replicates (ng/dl) Deviation Variation(%)
D10 29.7%0.995 3.4
E10 34.5%1.160 3.4
F10 41.6%1.151 2.8
3. Specificity
The monoclonal antibody used in the Atlas Link Thyroid Uptake Assay was evaluated for cross-reactivity using the Atlas Link T4 Kit. Results are expressed as the ratio of T4 concentration to the concentration of the cross-reactant that will displace 50% of the bound T4 enzyme conjugate x 100%
Cross-Reactant% Cross-Reactivity
I-Thyroxine (T4)(100%)
d-Thyroxine 86%
I-Triiodothyronine 1.2%
d-Triiodothyronine (T3) 0.6%
Diiodothyronine< 0.05%
Diiodotyrosine< 0.05%
Iodotyrosine< 0.05%
Phenytoin< 0.05%
BIBLIOGRAPHY
- Tietz, N.W., Fundamentals of Clinical Chemistry, 2nd Ed., pg. 602, Saunders Press, Phila., (1976).
- Robbins, J. Cheng S.Y. et al. Rec. Prog. Hormone Res. 7:307 (1977).
- Hamada, S., Nakagawa, T. et al. J. Clin. Endocrinol. Metab. 31:166 (1970).
- Nusynowitz, M.L. The Ligand Review 2:39 (1980).
- Sati, C., Chattor, A.J., Watts, N. In Fundamentals of Clinical Chemistry, Ed. Tietz, N. W. 3rd Edition, Pg. 586. Saunders Press, Phila. (1987).
- Ingbar, S.H. et al., J. Clin. Invest.(1965) 44:1679.
- Selenkow, H.A., and Robin, N.I.J. Maine Med. Assoc. (1970) 61:199
- Oppenheimer, J.H. et al., J. Clin.Invest. (1962) 42:1769
- Dick, M., Watson, F., Med J Aust. (1980) 1:115
- Dussault, J.H. Turcotte, R., and Gieyda, H., Journal of Clin Endocrin and Metabol. 1976, 42:232-285
- Tarnoky, A.L., Advances in Clinical Chem. (1981), 21:101
- Ruiz, M., et al., N.E.J. Med., (1982), 306:635.
Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
THYROID UPTAKE ENZYME IMMUNOASSAY KIT; Page 1
Atlas Link Thyroid Uptake EIA Procedure
Incubate Incubate
30 min 20 min
at room temp at room temp
Read
at
492 nm
25 L 100 L Decant & 100 L 100 L
Serum Enzyme Conjugate Wash (4x) Substrate Stopping
Reagent Reagent
Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664