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
http://www.atlaslink-inc.com, info@atlaslink-inc.com
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 thyroxinebinding 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.
Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
http://www.atlaslink-inc.com, info@atlaslink-inc.com
THYROID UPTAKE ENZYME IMMUNOASSAY KIT; Page 2
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 (2-8C) for 7 days. If storage time exceeds seven
days then frozen storage (-20C) 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
1.
2.
3.
4.
5.
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 37C 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.
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)
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
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
Euthyroid Calibrator.
(TU Ref=38%)
0.970
1.018
Hypothyroid Control
1.310
1.273
Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
http://www.atlaslink-inc.com, info@atlaslink-inc.com
%Uptake
29%
THYROID UPTAKE ENZYME IMMUNOASSAY KIT; Page 3
Hyperthyroid Control
a.
0.713
0.706
Do not mix or interchange reagent lots with any other kit or
different kit lots.
Do not use reagents beyond the expiration date jprinted on
each vial or bottle.
Each laboratory should establish its own criteria for
precision and accuracy by running controls in the
hypothyroid and hyperthyroid ranges.
Trend charts and statistical methods should be updated to
assure that performance is reliable and consistent from lot
to lot.
If the hypothyroid or hyperthyroid controls are not in their
established range, the test results may not be valid.
53%
b.
Patient 1
Patient 2
Patient 3
1.345
1.263
29%
c.
1.075
1.024
36%
d.
0.759
0.733
51%
e.
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).
EXPECTED VALUES
A.
Euthyroid
33-43 % Uptake
Hyperthyroid > 43 % Uptake
Hypothyroid < 33 % Uptake
B.
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).
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
Automation
30%
57%
13%
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:
Method
Correlation
Coefficient
RA/RIA
0.97
Slope
Intercept
0.96
- 0.03
2. Precision
a. Intra-assay Precision was determined by assaying 14
replicated of each of the three serum pools.
Pool
No of
Replicates
A
B
C
14
14
14
b.
LABORATORY QUALITY CONTROL
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
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.
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:
Mean
(ng/dl)
27.6%
33.4%
40.5%
Standard Coefficient of
Deviation Variation(%)
0.842
0.928
1.091
3.1
2.8
2.7
Inter-assay Precision was determined by assaying
duplicates of three serum pools in ten separate runs.
Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
http://www.atlaslink-inc.com, info@atlaslink-inc.com
THYROID UPTAKE ENZYME IMMUNOASSAY KIT; Page 4
Pool
No of
Replicates
D
E
F
10
10
10
Mean
(ng/dl)
29.7%
34.5%
41.6%
Standard Coefficient of
Deviation Variation(%)
0.995
1.160
1.151
3.4
3.4
2.8
< 0.05%
< 0.05%
< 0.05%
< 0.05%
BIBLIOGRAPHY
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
I-Thyroxine (T4)
d-Thyroxine
I-Triiodothyronine
d-Triiodothyronine (T3)
Diiodothyronine
Diiodotyrosine
Iodotyrosine
Phenytoin
% Cross-Reactivity
(100%)
86%
1.2%
0.6%
1. Tietz, N.W., Fundamentals of Clinical Chemistry, 2nd Ed.,
pg. 602, Saunders Press, Phila., (1976).
2. Robbins, J. Cheng S.Y. et al. Rec. Prog. Hormone Res.
7:307 (1977).
3. Hamada, S., Nakagawa, T. et al. J. Clin. Endocrinol. Metab.
31:166 (1970).
4. Nusynowitz, M.L. The Ligand Review 2:39 (1980).
5. Sati, C., Chattor, A.J., Watts, N. In Fundamentals of Clinical
Chemistry, Ed. Tietz, N. W. 3rd Edition, Pg. 586. Saunders
Press, Phila. (1987).
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
12. Ruiz, M., et al., N.E.J. Med., (1982), 306:635.
Atlas Link Thyroid Uptake EIA Procedure
Incubate
30 min
at room temp
Incubate
20 min
at room temp
Read
at

25 L
Serum

100 L
Enzyme Conjugate

Decant &
Wash (4x)

100 L
Substrate
Reagent
Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
http://www.atlaslink-inc.com, info@atlaslink-inc.com
492 nm
100 L
Stopping
Reagent