FREE T4 ENZYME IMMUNOASSAY TEST KIT; Page 1
Atlas Link
12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
Free T4 Enzyme Immunoassay Test Kit
FOR THE QUANTITATIVE DETERMINATION
OF FREE T4 IN SERUM
Cat. No . 3147
INTENDED USE
Free Thyroxine (FT4) Enzyme Immunoassay test kit is to be used for the in vitro determination of free thyroxine concentration inhuman serum.
EXPLANATION OF TEST
Thyroxine (T4) or 3,5’,5’-tetraiodothyronine is the most commonly measured thyroid hormone for the diagnosis of thyroid function. T4 has its primary influence on protein synthesis and oxygen consumption in virtually all tissues but it is also important for growth, development and sexual maturation.(1)
T4 is synthesized by the thyroid gland and is secreted into the bloodstream. Here the T4 becomes bound to serum proteins for transport to the cells. The major transport protein is Thyroxine Binding Globulin (TBG) which normally accounts for 80% of the bound T4. Other thyroid hormone binding proteins are Thyroxine Binding Prealbumin and Albumin. Most of the serum T4 is bound to these transport proteins leaving only about .03% free to exert its effect on cells. It is the Free T4 (FT4) that represents the metabolically active fraction; for this reason the measurement of Free T4 concentration is considered to be an indicator of patient thyroid status.
Primary hypothyroidism results in underproduction of T4 by the thyroid gland and consequently an abnormally low circulating FT4 concentration in the blood. Primary hyperthyroidism leads to excessive thyroid production of T4 and resulting elevated FT4 concentration.
Total serum T4 concentrations are dependent on the level of circulating TBG as well as the patient’s thyroid status. The concentration of TBG can be affected by certain drugs, steroid hormones, pregnancy, and by various nonthyroid illnesses. In an earlier generation of thyroid function tests, the effect of variable TBG concentration was dealt with by calculating a Free Thyroxine Index (FTI).(2) This FTI is the product of Total T4 concentration and Thyroid Uptake (TU), which assesses the number of available binding sites on the TBG. This approach requires carrying out two separate assay determinations (Total T4 and TU), but does provide a better indicator of thyroid status than Total T4 alone.
Free T4 tests are designed to directly reflect the equilibrium existing in serum between T4 and TBG-bound T4. These methods, including the FT4 test, can generally reflect thyroid status in a single assay.
Free T4 test is a rapid, sensitive method for measuring Free Thyroxine concentration in human serum. The method utilizes a highly specific anti-T4 monoclonal antibody bound to a solid support and a T4-labeled enzyme conjugate employed in a competitive immunoassay. After incubation, separation, and wash cycles, the absorbance of the colored end product of an enzyme-substrate reaction is measured by a microplate reader.
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 in both large and small laboratories.
PRINCIPLE OF THE METHOD
FT4 test is a solid phase competitive enzyme immunoassay. Patient serum, standards, and buffer are added to wells coated with a monoclonal T4 antibody. After an incubation, T4 labeled enzyme conjugate is added and incubation is continued. Free T4 in patient specimen and the wells are washed with buffer which removes any unbound T4-enzyme conjugate. The wells are then incubated with a substrate for the enzyme. The product of the enzyme-substrate reaction forms a colored complex when the stopping reagent is added. The absorbance is measured spectrophotometrically at 492 nm. A standard curve is prepared relating intensity of the color to the concentration of FT4. The concentration of FT4 in the patient sample is interpolated from the curve.
REAGENTS
Note: Store all reagents at 2-8 degree C
1. FT4 Serum Standard, (0 ng/dl), (1.0ml). Each vial contains freeze-dried human serum and .1% sodium azide as a preservative.
2. FT4 Serum Standard, (.2, .8, 2, 4, 8 ng/dl), (.5ml each). Each vial contains freeze-dried human serum, thyroxine, and .1% sodium azide as a preservative.
CAUTION: HANDLE AS IF CAPABLE OF TRANSMITTING HEPATITIS AND HUMAN IMMUNODEFICIENCY VIRUS. Source materials from which the standards were derived were found to be non-reactive for HBsAg and HIV when tested with licensed reagents. However, no known test method can assure that a product derived from human sources does not contain these viruses.
3. T4 Antibody Coated Wells, Each package contains 96 monoclonal T4 antibody-coated polystyrene wells.
4. Free T4 Enzyme Conjugate, (20ml or 200ml). Each bottle contains T4-labeled alkaline phosphatase, phosphate buffer, bovine serum albumin, and .1% sodium azide as a preservative.
5. Free T4 Assay Buffer, (20ml or 60ml). Each bottle contains phosphate-buffer, bovine serum albumin, and .1% sodium azide as a preservative.
6. 10x Wash Buffer Concentrate, (55ml or 200ml). Each bottle contains Tris-buffered saline, bovine serum albumin, magnesium chloride, and .1% sodium azide as a preservative.
7. Substrate Reagent, (20ml or 150ml). Each bottle contains carbonate buffer, magnesium chloride, aminoantipyrene, phenyl phosphate, and .1% sodium azide as a preservative.
8. Stopping Reagent, (20ml or 150ml). Each bottle contains phosphate buffer, potassium ferricyanide and .05% sodium azide as a preservative.
WARNING: Care should be taking in discarding the reagents containing sodium azide. Sodium Azide may react with lead and copper plumbing to form explosive metal azides. On disposal, flush with a large volume of water to prevent azide build-up.(13)
NOTE: DISPOSAL This kit contains material such as sodium azide and human serum which may necessitate special disposal procedures. Before disposing a leftover reagents, check local disposal regulations.
SPECIMEN COLLECTION AND HANDLING
1. Handle all blood and serum as if capable of transmitting Hepatitis and HIV.
2. Serum is required for this assay procedure.
3. Separate the serum immediately and store tightly capped.
4. Specimens may be stored refrigerated (2-8C) for 7 days. If storage time exceeds 7 days, store frozen (-20 C) for up to one month.
5. AVOID MULTIPLE FREEZE-THAW CYCLES.
6. Prior to assay, frozen sera should be completely thawed and mixed well.
7. DO NOT USE GROSSLY LIPEMIC SPECIMENS.
8. Moderately lipemic, hemolyzed, and icteric specimens will not interfere with the assay.
9. Sample degradation as well as multiple freeze-thaw cycles may cause inaccurate FT4 determinations. Patient specimens should be assayed as soon as possible.
MATERIALS NEEDED BUT NOT PROVIDED
1. Calibrated micropipettes (25, 100, 500, 1000 microliters).
2. Distilled/deionized water.
3. Absorbant paper.
4. Microwell strip or plate reading spectrophotometer, capable of reading at a wavelength of 490-510 nm.
5. Commercially available controls.
6. Beaker or flask for dilution of 10x Wash Buffer Concentrate.
7. Timer.
REAGENT PREPARATION
1. Free T4 Serum Standards. Reconstitute the 0 nl/dl standard) with 1.0 mL of distilled/deionized water. Reconstitute each of the remaining standards with .5 mL of distilled /deionized water. Replace cap and mixed gently until solution is fully homogenous. Store at 2-8 C; expiration date is on vial label.
2. 10x Wash Buffer Concentrate. Dilute the Wash Buffer Concentrate 1:10 with distilled/deionized water. To dilute the entire bottle, add the contents of the bottle to either 495 mL or 1800ml) of distilled/deionized water. The working dilution should be stored at 2-8 C. The expiration is equal to the expiration date on the concentrate label. Smaller quantities may be prepared by diluting 1 volume of 10x Wash Buffer Concentrate with 9 volumes of distilled/deionized water.
3. All other reagents are provided to use.
4. Prior to assay, warm all reagents to ambient temperature by allowing them to stand at room temperature, or by briefly warming them in a 37 C water bath. Gently mix all reagents.
PROCEDURAL NOTES
1. It is recommended that standards, controls and samples be run in duplicate.
2. Optimal results will be obtained by strict adherence to this protocol. Accurate and precise pipetting, as well as following the exact time and temperature requirement prescribed, are essential. Any deviation from this may yield invalid data.
3. When pipetting 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.
4. Read the absorbances within one hour after completing the assay.
5. Controls should be run with every assay.
6. If the microwell reading spectrophotometer requires a reagent blank, mix 100 l of Substrate Reagent with a 100 l of Stopping Reagent in an unused well. Zero or blank instrument with this well.
7. 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.
8. Take special care that the strips do not fall out of the holder when decanting.
FREE T4 ASSAY PROCEDURE
Review procedural notes prior to the performing assay.
1. Pipet 25 l of FT4 standards into the appropriate wells. (The 0 nl/dl standard must be run if using a previously stored curve).
2. Pipet 25 l of each control and patient serum into the appropriate wells.
3. Pipet 100 l of FT4 Assay Buffer into all wells.
4. Incubate for 30 minutes at room temperature (18-26 C)
5. Pipet 100 l FT4-Enzyme Conjugate into all wells.
6. Incubate for 10 minutes at room temperature (18-26 C)
7. Decant or aspirate all wells. Fill wells with prepared Washed Buffer. Decant or aspirate. Wash the wells 3 more times in this manner. Aspirate or blot on absorbant paper to remove all liquid from wells.
8. Add 100 l of the Substrate Reagent to all wells.
9. Incubate for 30 minutes at room temperature (18-26 C)
10. Add 100 l of Stopping Reagent to all wells.
11. Read absorbance on Microwell Reader at 490-510 nm. All wells must be read within 1 hour of assay completion. (If available, follow the Instrument Application Method for the instrumentation used.)
RESULTS
The standard curve may be constructed using two different methods. The results obtained by either method are identical.
Method 1: Absorbance Plot
1. To construct the standard curve, plot the absorbance for the FT4 standards (vertical axis) on the semi-logarithmic graph paper supplied.
2. Draw the best curve through the points taken as a set.
3. Using the standard curve, interpolate the control and unknown serum values from each absorbance measured. Record the value for each control or unknown sample.
Method 2: Normalized % Absorbance Plot
1. Divide all absorbance by the absorbance value for 0 ng/dl standard level and multiply by 100%
% A/Ao= A x 100
Ao
where:
A = average absorbance for standard, controls and patients samples
Ao = average absorbance value for 0 ng/dl standard.
2. Construct the standard curve by plotting the % A/Ao values for the FT4 standards (vertical axis) versus the FT4 standard concentrations (horizontal axis) on the graph paper provide.
3. Draw a best fit curve through the points taken as a set.
4. Interpolate the control and serum values from each % A/Ao value obtained.
CAUTION: If control values deviate from their established range the assay is not valid.
OPTIONAL ASSAY PROCEDURE USING SINGLE POINT CALIBRATION CURVE
For individual samples or low volume usage, the following optional procedure may be employed.
I. Initial Calibration Curve
1. For each new kit lot run one complete standard calibration curve as described in the Assay Procedure Section.
2. Construct a standard curve as described in Method 2 above.
3. Save this calibration curve for use in all subsequent runs using only single point calibration.
II. Subsequent Assays
1. Run the 0 ng/dl standard, controls and patient samples according to the assay protocol.
2. Calculate %A/Ao as described above.
3. Using the original standard calibration curve, interpolate the control and patient values.
CAUTION: If control values deviate from their established range the assay is not valid.
REPRESENTATIVE DATA
FT4 Standard or Sample Absorbance %A/Ao FT4 Conc
0.0 ng/dl1.240
1.249
0.2 ng/dl1.134 90%
1.105
0.8 ng/dl0.856 70%
0.877
2.0 ng/dl0.544 43%
0.536
4.0 ng/dl0.304 23%
0.089
Sample 11.050 83% 0.39 ng/dl
1.013
Sample 20.680 54% 1.43 ng/dl
0.660
Sample 3 0.310 25% 3.7 ng/dl
0.322
EXPECTED VALUES
It is recommended that each laboratory establish its own normal ranges based on a representative sampling of the local population.
Hypothyroid: less than 0.85 ng/dl
Euthyroid: 0.85 - 1.85 ng/dl
Hyperthyroid: greater than 1.85 ng/dl
CLINICAL SIGNIFICANCE
Alterations in the concentration of serum binding proteins will generally result in a corresponding change in Total T4 concentrations while the physiologically active Free T4 level remains largely unchanged in a euthyroid individual. Therefore, determination of Free T4 concentration may provide a more accurate assessment of thyroid status than Total T4 measurement. Elevated Free T4 concentrations are indicative of hyperthryoidism and low levels are indicative of hypthyroidism.
LABORATORY QUALITY CONTROL
1. Do not mix or interchange reagents lots with any other kit or different kit lots.
2. Do not use reagents beyond expiration date imprinted on each vial or bottle label.
3. Each laboratory should establish its own criteria for precision and accuracy by running controls in the normal range, as well as in the low and elevated ranges.
4. Trend charts and statistical methods should be updated to ensure that performance is reliable and consistent from lot to lot.
LIMITATION OF THE PROCEDURE
It has been determined that FT4 levels correlate well with the thyroid status of the patient. However, there are situations that can make the interpretation of FT4 results complex and FT4 measurements should not be the sole basis for determining thyroid status. Even the most reliable FT4 measurement may not correct for extreme variation is serum binding proteins and other factors.
Normal thyroid hormone levels do not exclude thyroid disease, and diffuse or nodular thyroid enlargements may be seen in euthyroid patients.(3)
A variety of causes apart from thyroid malfunction may give rise to abnormal FT4 concentrations in serum
In patients receiving heparin therapy, the equilibrium between Free T4 and TBG bound T4 may be affected.(4) Heparin therapy is known to increase the concentration of non-esterified fatty acids which may displace T4 on serum binding proteins and thus cause an elevation of Free T4 values. Phenytoin, salycilate and other drugs can also interfere with the binding of T4 to TBG(5) in much the same way. One must be cautious when interpreting Free T4 results patients being treated with heparin or other drugs which affect binding of T4 to serum proteins.
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 FT4 levels.
Familial dysalbuminemic hyperthyroxemia(9), auto-antibodies to T4(10) and analbuminemia can result in elevation of Total T4 but should not affect FT4 levels unless the patients are receiving T4 treatment. (10)
The treatment of hypothyroidism with L-thyroxine can result in lack of correlation between clinical status and FT4. The FT4 concentration can be elevated in these cases.(12)
Severely hemolyze, lipemic, or icteric samples may result in inaccurate values. A better quality specimen should be obtained if possible.
PERFORMANCE CHARACTERISTIC
1. Correlation with Radioimmunoassay
A total of 144 serum standards were run in the FT4 procedure and in a commercially available RIA procedure. The following correlation was obtained.
Correlation CoefficientSlopeIntercept
0.920.97- 0.01
2 Precision
a. Intra-assay Precision was determined by assaying 12 replicated of each of the three serum pools.
PoolNo of MeanStandard Coefficient of
Replicates (ng/dl) Deviation Variation (%)
D12 1.0120.0747.4
E12 1.1920.0675.6
F12 2.8750.1224.2
b. Inter-assay Precision was determined by assaying duplicates of 3 serum pools in 10 separate runs, using a standard curve concstructed for each run.
PoolNo of MeanStandard Coefficient of
Replicates (ng/dl) Deviation Variation (%)
D10 0.2730.0155.5
E10 1.1500.0716.2
F10 2.1300.1487.0
3. Sensitivity
The sensitivity of the method is defined as the lowest concentration of FT4 can be distinguished from 0 ng/dl as calculated from the 95% confidence limits of the 0 ng/dl standard absorbance. This method will reliably detect FT4 concentration as low as 0.04 ng/gl
4. Specificity
Results are expressed as the ratio T4 concentration to the concentration of the cross-reactant that will displace 50% of the bound T4 enzyme.
Cross-Reactant% Cross-Reactivity
I-Thyroxine (T4)(100)
d-Thyroxine 100
I-Triiodothyronine (T4)4.5
d-Triiodothyronine (T3)5.4
Diiodothyronine< 0.06
Diiodotyrosine< 0.05
Iodotyrosine< 0.05
Phenytoin< 0.05
Sodium Salicylate< 0.05
5. Effect of Exogenous Added Drugs
Free T4 concentration were determined in the presence of each of several drugs added to serum at approximately twice the normal therapeutic serum concentration level. The % change in apparent Free T4 found in the presence of these drugs was measured, and is shown in the table below.
SubstanceFinal Concentration% Change in T4
Exogenous DrugConcentration
Sodium Salicylate 40 mg/dl + 1.8
Diphenylhydantoin 30 g/dl + 28.0
Propylthiouracil 4 mg/dl + 18.7
Phenylbutazone 30 g/dl + 0.8
6. Effects of Endogenous Estrogens
Samples from 3rd trisemester pregnant women were analyzed to determine thyroid status by assaying for Total T4, Thyroid Uptake, and Free T4. In 33% of the samples, the Total T4 levels were elevated to borderline hyperthyroid; 67% were eurothyroid. The calculated Free Thyroxine index and Free T4 concentration were found to be well within the normal range for all of the samples.
REFERENCES
1. Tietz, N.W., Fundamentals of Clinical Chemistry, 2nd Ed., pg. 602, Saunders Press, Phila., 1976.
2. Horworth, P.J.N., Ward, RL., J. Clin Pathol. 1972;25;259-62.
3. Sati, C., Chattor, A.J., Watts, N. In Fundamentals of Clinical Chemistry, Ed. Tietz, N,. W. 3rd Edition, Pg. 586. Saunders Press Phila. 1987.
4. Lundberg, P.A. Jagenburg, R., Lindstedt, G., Nystrom, E., Clin. Chem. 1982, 28:1241.
5. Melmed, S., Geola, F.L., Reed, A.W., Pekary, A.E., Park, J. Hershmen, J.M. Clin Endocrin and Metabol. 1982, 54:300.
6. Ingbar, S.H. et al. J. Clin. Invest., 1965 44:1679.
7. Selenkow, H.A. and Robin, N.I.J. Maine Med. Assoc 1970 61:199
8. Oppenheimer, J.H. et al. J. Clin.Invest. 1962 42:1769
9. Dick, M., Watson, F., Med J Aust. 1980 1:115
10. Dussault, J.H. Turcotte, R., and Gieyda, H., Journal of Clin Endocrin and Metabol. 1976, 42:232-285
11. Tarnoky, A.L. Advances in Clinical Chem. 1981, 21:101-146
12. Emrich D., Schondube, H. Sehlen, S., and Schreivagel, I. Nuc. Compact, 1985, 16:392
13. Procedures for Decontamination of Plumbing System Containing Copper and/or Lead Azides, Dept of. H.E.W., N.I.O.S.H., Rockville Maryland, 1976
FT4 Assay Procedure