AgAbC.W.
+
EnzAgAbC.W.rev: February 4, 2000
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Intended Use: The Quantitative Determination of Free Thyroxine
Concentration in Human Serum by a Microplate Enzyme Immunoassay. Levels of
fT4 are thought to reflect the amount of T4 available to the cells and may
therefore determine the clinical metabolic status of T4.
Thyroxine, the principal thyroid hormone,
circulates in blood almost completely bound to carrier proteins. The main
carrier is thyroxine-binding globulin (TBG). However, only the free (unbound)
portion of thyroxine is 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 thyroxine level changes so that the free
thyroxine concentration remains constant. Thus, measurements of free thyroxine
concentrations correlate better with clinical status than total thyroxine
levels.
For example, the increase in total thyroxine
associated with pregnancy, oral contraceptives and estrogen therapy occasionally
result in total T4 levels over the limits of normal while the free thyroxine
concentration remains in the normal reference range. Masking of abnormal
thyroid function can also occur in both hyper and hypothyroid conditions
by alterations in the TBG concentration. The total T4 can be elevated or
lowered by TBG changes such that the normal reference levels result. Again,
the free thyroxine concentration typically uncovers the patient’s actual
clinical status.
This microplate enzyme immunoassay methodology
provides the technician with optimum sensitivity while requiring few technical
manipulations. In this method,
serum reference, patient specimen, or control is first added to a microplate
well. Enzyme-T4 conjugate (analog
method) is added, then the reactants are mixed. A competition reaction results
between the enzyme conjugate and the free thyroxine for a limited number
of antibody combining sites immobilized on the well.
After the completion of the required incubation
period, the antibody bound enzyme-thyroxine conjugate is separated from the
unbound enzyme-thyroxine 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 thyroxine 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 thyroxine
concentration.
The essential reagents required for a solid
phase enzyme immunoassay include immobilized antibody, enzyme-antigen conjugate
and native antigen.
Upon mixing immobilized antibody, enzyme-antigen
conjugate and a serum containing the native free antigen, a competition reaction
results between the native free antigen and the enzyme-antigen conjugate
for a limited number of insolubulized binding
sites. The interaction is
illustrated by the followed equation:
ka
EnzAg
+
Ag
+
AbC.W.
AgAbC.W.
+
EnzAgAbC.W.
k-a
AbC.W. = Monospecific Immobilized Antibody (Constant
Quantity)
Ag = Native Antigen (Variable
Quantity)
EnzAg = Enzyme-antigen Conjugate (Constant
Quantity)
AgAbC.W. = AntigenAntibody Complex
EnzAg
AbC.W. =
Enzyme-antigen Conjugate Antibody Complex
ka
= Rate Constant of
Association
ka = Rate Constant
of Disassociation
K = ka / k-a = Equilibrium
Constant
After equilibrium is attained, the
antibodybound fraction is separated from unbound antigen by decantation
or aspiration. The enzyme activity
in the antibodybound fraction is inversely proportional to the native free
antigen concentration. By utilizing
several different serum references of known antigen concentration,
a dose response curve can be generated from which the antigen concentration
of an unknown can be ascertained.
REAGENTS
FOR 96-well MICROPLATE
A.
Human Serum References -- 1.0
ml/vial - Icons A-F
Six (6) vials of serum reference for thyroxine
at approximate* concentrations of 0
(A), 0.3
(B), 0.95
(C), 2.1 (D), 3.6
(E) and 7.0
(F) ng/dl. Store at
28°C.
A preservative has been added. * Exact levels are given on the labels
on a lot specific basis.
For SI units: 1ng/dl x 12.9 =
pmol/L
B. fT4- Enzyme Reagent –
10.5ml/vial - Icon
One (1) vial of thyroxine-horseradish peroxidase (HRP) conjugate
in a bovine albumin-stabilizing matrix. A preservative has been added. Store
at
28°C.
C.
T4 Antibody Coated Microplate
-- 96 wells -
Icon
One 96-well microplate coated with sheep
anti-thyroxine serum and packaged in an aluminum bag with a drying
agent. Store at
28°C.
D. Wash Solution Concentrate
-- 20ml - Icon
One (1) vial containing a surfactant in phosphate
buffered saline. A preservative has been added. Store at
2-30°C.
E.
Substrate A --7.0ml/vial
- Icon SA
One (1) bottle containing tetramethylbenzidine (TMB) in
buffer. Store at
28°C.
F.
Substrate B -- 7.0ml/vial -
Icon SB
One (1) bottle containing hydrogen peroxide
(H2O2) in buffer.
Store at
28°C.
G.
Stop Solution -- 6.0ml/vial
- Icon
One (1) bottle containing a strong acid (1N
HCl). Store at
230°C.
Note
1:
Do
not use reagents beyond the kit expiration date.
Note
2:
Opened
reagents are stable for sixty (60) days when stored at
28°C.
PRECAUTIONS
For
In Vitro Diagnostic
Use
Not
for Internal or External Use in Humans or
Animals
All products that contain human serum have been found to be
non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV Antibodies
by FDA required tests. Since
no known test can offer complete assurance that infectious agents are absent,
all human serum products should be handled as potentially hazardous and capable
of transmitting disease.
Good laboratory procedures for handling blood products can be found
in the Center for Disease Control / National Institute of Health, "Biosafety
in Microbiological and Biomedical Laboratories," 2nd Edition, 1988, HHS
Publication No. (CDC) 88-8395.
SPECIMEN
COLLECTION AND PREPARATION
The specimens shall be blood serum in type
and the usual precautions in the collection of venipuncture samples should
be observed. For accurate comparison to established normal values, a fasting
morning serum sample should be obtained. The blood should be collected in
a plain red-top venipuncture tube without additives or gel barrier. Allow
the blood to clot. Centrifuge the specimen to separate the serum from the
cells.
The crossreactivity of the thyroxine antibody
to selected substances was evaluated by adding the interfering substance
to a serum matrix at various concentrations. The cross-reactivity was calculated
by deriving a ratio between dose of interfering substance to dose of
thyroxine needed to displace the same amount of
tracer.
l–Thyroxine
1.0000
----
d-Thyroxine
0.9800
10µg/dl
dTriiodothyronine
0.0150 100µg/dl
l–Triiodothyronine
0.0300
100µg/dl
lodothyrosine
0.0001
100µg/ml
Diiodothyrosine
0.0001
100µg/ml
Diiodothyronine
0.0001
100µg/ml
MATERIALS
Provided:
1. Six
(6) vials of free thyroxine human serum references.
2. One
(1) vial of thyroxine-enzyme reagent.
3. One
96-well T4 antibody coated
microplate.
4. One
(1) bottle of wash buffer concentrate.
5. One
(1) bottle of Substrate A.
6 One
(1) bottle of Substrate B.
7. One
(1) bottle of Stop solution.
8.
Instructions.
Required
But Not
Provided:
1. Pipette
capable of delivering 50µl volumes with a precision of better than
1.5%.
2.
Dispenser(s) for repetitive deliveries of 0.100ml and 0.300ml volumes
with a precision of better than 1.5%.
3.
Adjustable volume (200-1000µl) dispenser(s) for substrate dilutions.
4.
Microplate washer or a squeeze bottle (optional).
5. Microplate Reader with 450nm
and 620nm wavelength absorbance capability.
6. Test tube(s) for mixing
substrates A&B.
7.
Absorbent Paper for blotting the microplate wells.
8. Plastic
wrap or microplate cover for incubation steps.
9. Vacuum
aspirator (optional) for wash steps.
10.
Timer.
11.
Quality control materials.
REAGENT
PREPARATION:
1.
Wash
Buffer
Dilute contents of Wash
concentrate to 1000ml with distilled or deionized water
in a suitable storage container. Store at room temperature until expiration
date printed on concentrate label. It
is essential that all the contents of the wash buffer concentrate
dissolve. Crystal formation
in the Wash Concentrate can be eliminated by briefly (approx. 5 minutes)
heating in a water bath at
37°C
or storing the Wash Concentrate at room
temperature.
2.
Working
Substrate Solution – Prepare daily
Determine the amount of reagent needed and
prepare by mixing equal portions of Substrate A and Substrate B in a suitable
container. For example, add 1ml of A and 1ml of B per two (2) eight well
strips (A slight excess of solution is made).
Use within twenty-four hours of
preparation for maximum performance of the assay.
TEST
PROCEDURE
Before
proceeding with the assay, bring all reagents, serum references and controls
to room temperature (20 -
27°C).
1. Format
the microplates’ wells for each serum reference, control and patient
specimen to be assayed in duplicate.
Replace any unused microwell strips back into the aluminum bag, seal and
store at
2-8°C
2. Pipette
0.050 ml (50µl) of the appropriate serum reference, control or specimen
into the assigned well.
3. Add
0.100 ml (100µl) of fT4-enzyme conjugate solution to all
wells.
4. Swirl
the microplate gently for 20-30 seconds to mix and cover.
5. Incubate
60 minutes at room
temperature.
6. Discard
the contents of the microplate by decantation or aspiration. If decanting,
blot the plate dry with absorbent paper.
7. Add
300µl of wash buffer (see Reagent Preparation Section), decant (tap
and blot) or aspirate. Repeat two (2) additional times for a total of three
(3) washes. An automatic or manual
plate washer can be used. Follow the manufacturer’s instruction for
proper usage. If a squeeze bottle is employed, fill each well by depressing
the container (avoiding air bubbles) to dispense the wash. Decant the wash
and repeat two (2) additional times.
8. Add
0.100 ml (100µl) of working substrate solution to all wells (see Reagent
Preparation Section). Always add reagents
in the same order to minimize reaction time differences between wells.
9. Incubate
at room temperature for fifteen (15) minutes.
10. Add
0.050ml (50µl) of stop solution to each well and gently mix for 15-20
seconds. Always add reagents in the
same order to minimize reaction time differences between wells.
11. Read
the absorbance in each well at 450nm (using a reference wavelength of 620-630nm
to minimize well imperfections) in a microplate reader.
The results should be read within thirty (30) minutes of adding the
stop solution.
13
Unknown #1
1.783
14 Unknown
#1
1.676
1.730
1.3 ng/dl
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
to follow the performance of the supplied reagents. Pertinent statistical
methods should be employed to ascertain trends. The individual laboratory
should set acceptable assay performance limits. Other parameters that should
be monitored include he 80, 50 and 20% intercepts of the standard curve for
run-to-run reproducibility. In addition, maximum absorbance should be consistent
with past experience. Significant deviation from established performance
can indicate unnoticed change in experimental conditions or degradation of
kit reagents. Fresh reagents
should be used to determine the reason for the
variations.
A dose response curve is used to ascertain
the concentration of free thyroxine in unknown
specimens.
1.
Record the absorbance obtained from the printout of the microplate
reader as outlined in Example 1.
2.
Plot the absorbance for each duplicate serum reference versus the
corresponding free T4 concentration in ng/dl 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 fT4 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 ng/dl) from the horizontal axis of the graph (the duplicates of the unknown
may be averaged as indicated). In the following example, the average absorbance
1.123 (intersects the standard curve at (8.1 ng/dl) fT4 concentration (See
Figure 1).
B.
Interpretation
If computer controlled data reduction is used
to interpret the results of the test, it is imperative that the predicted
values for the calibrators fall within 10% of the assigned
concentrations.
Total serum thyroxine concentration is dependent
upon a multiplicity of factors: thyroid gland function and its regulation,
thyroxine binding globulin (TBG) concentration, and the binding of thyroxine
to TBG (3, 4). Thus, total thyroxine
concentration alone is not sufficient to assess clinical
status.
Total serum thyroxine values may be
elevated under conditions such as pregnancy or administration of oral
contraceptives. A T3 uptake test may be performed to estimate the relative
TBG concentration in order to determine if the elevated T4 is caused by TBG
variation.
A decrease in total thyroxine values is found
with proteinwasting diseases, certain liver diseases and administration
of testosterone, diphenylhydantoin or
salicylates. A table of interfering drugs and conditions which affect
total thyroxine values has been compiled by the Journal of the American
Association of Clinical Chemists.
"NOT
INTENDED FOR NEWBORN
SCREENING"
EXAMPLE
1
1
0.0
ng/dl
2.462
2
0.0
ng/dl
2.531
3
0.3
ng/dl
2.330
4
0.3
ng/dl
2.255
5
0.95
ng/dl
1.915
6
0.95
ng/dl
1.892
7
2.1
ng/dl
1.328
8
2.1
ng/dl
1.262
9
3.6
ng/dl
0.834
10
3.6
ng/dl
0.804
11
7.0
ng/dl
0.399
12
7.0
ng/dl
0.421
Unknown
Avg.
WELL
I. D.
O.D.
O.D.
Value
 |
*The data presented in
Example 1 and Figure 1 are for illustration only and
should not be used in lieu of a standard curve prepared with
each assay.
Q.C.
PARAMETERS
Maximum
Absorbance (O calibrator) = > 1.2
LIMITATIONS
OF PROCEDURE
A. Assay
Performance
It is important that the time of reaction
in each well is held constant for reproducible results. Pipetting of samples
should not extend beyond ten (10) minutes to avoid assay drift. If more than
one (1) plate is used, it is recommended to repeat the dose response
curve.
Addition of the substrate solution initiates
a kinetic reaction, which is terminated by the addition of the stop solution.
Therefore, the addition of the substrate and the stopping solution should
be added in the same sequence to eliminate any time deviation during
reaction.
Plate readers measure vertically. Do not touch
the bottom of the wells.
Failure to remove adhering solution adequately
in the aspiration or decantation wash step(s) may result in poor replication
and spurious results.
Sample(s), which are contaminated
microbiologically, should not be used in the assay. Highly lipemeic or hemolysed
specimen(s) should similarly not be used
EXPECTED
RANGES OF VALUES
A study of euthyroid adult population was
undertaken to determine expected values for the Free T4 EIA Test
System. The mean (R) values,
standard deviations
(s) and expected ranges (±2
s) are presented in Table
1.
TABLE
I
Expected
Values for the Free T4 EIA Test
System
(in
ng/dl)
(110
specimens)
(30specimens)
Mean
(X)
1.4
1.5
Standard Deviation
(s)
0.6
0.7
Expected Ranges (±2
s)
0.8 – 2.0 0.8 –
2.2
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
analyst. For these reasons each
laboratory should depend upon the range of expected values established by
the Manufacturer only until an inhouse range can be determined by the analysts
using the method with a population indigenous to the area in which the laboratory
is located.
TABLE
2
Within
Assay Precision (Values in ng/dl )
Sample
N
X
S.D.
C.V.
Low
16
0.3
0.03
9.8%
Normal
16
1.4
0.06
4.5%
High
16
3.6
0.22
6.2%
TABLE
3
Between
Assay Precision (Values in ng/dl )
Sample
N
X
s
C.V.
Low
10
0.34
0.04
11.5%
Normal
10
1.35
0.07
3.7%
High
10
3.69
0.25
4.2%
*As measured in ten experiments in duplicate
over a ten day period.
B. Accuracy
The T4 Microplate EIA Test System was compared
with a coated tube radioimmunoassay
method. Biological specimens
from hypothyroid, euthyroid and hyperthyroid populations were used (The values
ranged from 0.1ng/dl – 8ng/dl). The total number of such specimens was
85. The least square regression
equation and the correlation coefficient were computed for this fT4 EIA in
comparison with the reference method.
The data obtained is displayed in Table 4.
Least
Square
Regression
Correlation
This
Method
1.5
y =
0.10+0.952(x) 0.978
Reference
1.4
Only slight amounts of bias between this method
and the reference method are indicated by the closeness of the mean
values. The least square regression
equation and correlation coefficient indicates excellent method
agreement.
C.
Sensitivity
The thyroxine procedure has a sensitivity
of 0.05 ng/dl. The sensitivity was ascertained by determining the variability
of the 0 ng/dl serum calibrator and using the
2s (95% certainty) statistic to calculate the
minimum dose.
REFERENCES
1. Barker,
S.B., "Determination of Protein Bound
Iodine.",Joumal Biological Chemistry,
173, 175,
(1948).
2. Chopra, I.J., Solomon, D.H.,
and Ho, R.S., "A Radioimmunoassay of Thyroxine",
J. Clinical EndocrinoL,
33, 865
(1971).
3. Young, D.S., Pestaner, L.C.,
and Gilberman, U., "Effects of Drugs on Clinical Laboratory Tests",
Clinical Chemistry,
21, 3660 (1975).
4. Sterling, L., Diagnosis and
Treatment of Thyroid Disease, Cleveland , CRC Press, P. 1951
(1975).
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USA
or 408-780-0908
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