3. Microplate washers or a squeeze bottle (optional). 2. Pipette 0.

050 ml (50µl) of the appropriate serum reference,

3.0 PRINCIPLE 4. Microplate Reader with 450nm and 620nm wavelength control or specimen into the assigned well.
absorbance capability. 3. Add 0.100 ml (100µl) of fT4 Enzyme Reagent to all wells.
Competitive Enzyme Immunoassay, Analog Method for Free-T4 5. Absorbent Paper for blotting the microplate wells. 4. Swirl the microplate gently for 20-30 seconds to mix and
(TYPE 5): 6. Plastic wrap or microplate cover for incubation steps. cover.
The essential reagents required for a solid phase enzyme 7. Vacuum aspirator (optional) for wash steps. 5. Incubate 60 minutes at room temperature.
immunoassay include immobilized antibody, enzyme-antigen 8. Timer. 6. Discard the contents of the microplate by decantation or
conjugate and native antigen. Upon mixing immobilized antibody, 9. Quality control materials. aspiration. If decanting, blot the plate dry with absorbent
enzyme-antigen conjugate and a serum containing the native free paper.
antigen, a competition reaction results between the native free 5.0 PRECAUTIONS 7. Add 350µl of wash buffer (see Reagent Preparation Section),
antigen and the enzyme-antigen conjugate for a limited number of decant (tap and blot) or aspirate. Repeat two (2) additional
insolubilized binding sites. The interaction is illustrated by the For In Vitro Diagnostic Use times for a total of three (3) washes. An automatic or manual
followed equation: Not for Internal or External Use in Humans or Animals plate washer can be used. Follow the manufacturer’s
ka All products that contain human serum have been found to be instruction for proper usage. If a squeeze bottle is
EnzAg + Ag + Ab AgAb C.W. + EnzAgAb C.W. non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV employed, fill each well by depressing the container
C.W.
k -a Antibodies by FDA licensed reagents. Since no known test can (avoiding air bubbles) to dispense the wash. Decant the
Ab C.W. = Monospecific Immobilized Antibody (Constant Quantity) offer complete assurance that infectious agents are absent, all wash and repeat two (2) additional times.
Ag = Native Antigen (Variable Quantity) human serum products should be handled as potentially 8. Add 0.100 ml (100µl) of working substrate solution to all wells
Enz
Ag = Enzyme-antigen Conjugate (Constant Quantity) hazardous and capable of transmitting disease. Good (see Reagent Preparation Section). Always add reagents in
Free Thyroxine (Free T4) Test AgAb C.W. = Antigen-Antibody Complex laboratory procedures for handling blood products can be found the same order to minimize reaction time differences
Enz
Ag Ab C.W. = Enzyme-antigen Conjugate -Antibody Complex in the Center for Disease Control / National Institute of Health, between wells.
System K a = Rate Constant of Association "Biosafety in Microbiological and Biomedical Laboratories," 2nd DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITION
Edition, 1988, HHS Publication No. (CDC) 88-8395. 9. Incubate at room temperature for fifteen (15) minutes.
Product Code: 1225-300 k -a = Rate Constant of Disassociation
Safe Disposal of kit components must be according to local 10. Add 0.050ml (50µl) of stop solution to each well and gently
K = k a / k -a = Equilibrium Constant
regulatory and statutory requirement. mix for 15-20 seconds. Always add reagents in the same
After equilibrium is attained, the antibody-bound fraction is order to minimize reaction time differences between wells.
separated from unbound antigen by decantation or aspiration. 6.0 SPECIMEN COLLECTION AND PREPARATION
1.0 INTRODUCTION 11. Read the absorbance in each well at 450nm (using a
The enzyme activity in the antibody-bound fraction is inversely reference wavelength of 620-630nm to minimize well
proportional to the native free antigen concentration. By utilizing The specimens shall be blood, serum in type and the usual imperfections) in a microplate reader. The results should be
Intended Use: The Quantitative Determination of Free several different serum references of known antigen con- precautions in the collection of venipuncture samples should be read within thirty (30) minutes of adding the stop solution.
Thyroxine Concentration in Human Serum by a Microplate centration, a dose response curve can be generated from which observed. For accurate comparison to established normal values, 10.0 CALCULATION OF RESULTS
Enzyme Immunoassay the antigen concentration of an unknown can be ascertained. a fasting morning serum sample should be obtained. The blood
A dose response curve is used to ascertain the concentration
should be collected in a plain redtop venipuncture tube without
2.0 SUMMARY AND EXPLANATION OF THE TEST 4.0 REAGENTS of free T4 in unknown specimens.
additives or anti-coagulants. Allow the blood to clot. Centrifuge
1. Record the absorbance obtained from the printout of the
the specimen to separate the serum from the cells.
Thyroxine, the principal thyroid hormone, circulates in blood Materials Provided: microplate reader as outlined in Example 1.
almost completely bound to carrier proteins. The main carrier is A. Free T4 Calibrators – 1 ml/vial - Icons A-F Samples may be refrigerated at 2-8oC for a maximum period of 2. Plot the absorbance for each duplicate serum reference
thyroxine-binding globulin (TBG). However, only the free Six (6) vials of human serum based reference calibrators for five (5) days. If the specimen(s) cannot be assayed within this versus the corresponding Free T4 concentration in ng/dl on
(unbound) portion of thyroxine is responsible for the biological free thyroxine at approximate* concentrations of 0 (A), 0.40 o linear graph paper (do not average the duplicates of the
time, the sample(s) may be stored at temperatures of -20 C for up
action. Further, the concentrations of the carrier proteins are (B), 1.25 (C), 2.10 (D), 5.00 (E) and 7.40 (F) ng/dl. Store at to 30 days. Avoid use of contaminated devices. Avoid repetitive serum references before plotting).
altered in many clinical conditions, such as pregnancy. In normal 2-8°C. A preservative has been added. For SI units use the freezing and thawing. When assayed in duplicate, 0.100ml of the 3. Connect the points with a best-fit curve.
thyroid function as the concentrations of the carrier proteins conversion factor 12.9 to convert ng/dl to pmol/L. specimen is required. 4. To determine the concentration of Free T4 for an unknown,
alters, the total thyroxine level changes so that the free thyroxine * Exact levels are given on the labels on a lot specific basis. locate the average absorbance of the duplicates for each
concentration remains constant. Thus, measurements of free 7.0 QUALITY CONTROL unknown on the vertical axis of the graph, find the
thyroxine concentrations correlate better with clinical status than B. fT4- Enzyme Reagent – 13 ml/vial - Icon E intersecting point on the curve, and read the concentration
total thyroxine levels. One (1) vial of thyroxine-horseradish peroxidase (HRP) Each laboratory should assay controls at levels in the (in ng/dl) from the horizontal axis of the graph (the
conjugate in a protein-stabilized matrix. A preservative has hypothyroid, euthyroid and hyperthyroid range for monitoring duplicates of the unknown may be averaged as indicated). In
The increase in total thyroxine associated with pregnancy, oral been added. Store at 2-8°C. assay performance. These controls should be treated as the following example, the average absorbance (0.964)
contraceptives and estrogen therapy occasionally result in total C. Free T4 Antibody Coated Plate – 96 wells - Icon unknowns and values determined in every test procedure intersects the dose response curve at (1.65ng/dl) free T4
T4 levels over the limits of normal while the free thyroxine One 96-well microplate coated with anti-thyroxine serum and performed. Quality control charts should be maintained to follow concentration (See Figure 1).
concentration remains in the normal reference range. Masking of packaged in an aluminum bag with a drying agent. Store at the performance of the supplied reagents. Pertinent statistical *The data presented in Example 1 and Figure 1 is for illustration
abnormal thyroid function can also occur in both hyper and 2-8°C. methods should be employed to ascertain trends. Significant only and should not be used in lieu of a standard curve prepared
hypothyroid conditions by alterations in the TBG concentration. D. Wash Solution Concentrate – 20ml - Icon deviation from established performance can indicate unnoticed with each assay. Assigned values for calibrators are lot
The total T4 can be elevated or lowered by TBG changes such One (1) vial containing a surfactant in buffered saline. A change in experimental conditions or degradation of kit reagents. specific.
that the normal reference levels result. The free thyroxine Fresh reagents should be used to determine the reason for the
preservative has been added. Store at 2-8°C. Figure 1
concentration can help in uncovering the patient’s actual clinical A variations.
status. E. Substrate A – 7 ml/vial - Icon S
8.0 REAGENT PREPARATION:
One (1) bottle containing tetramethylbenzidine (TMB) in 3.000
1. Wash Buffer 2.500
In this method, serum reference, patient specimen, or control is acetate buffer. Store at 2-8°C. A
B Dilute contents of wash concentrate to 1000ml with distilled or b 2.000
first added to a microplate well. Enzyme-T4 conjugate (analog F. Substrate B – 7 ml/vial - Icon S s
deionized water in a suitable storage container. Diluted buffer o
1.500
method) is added and the reactants are mixed. A competition One (1) bottle containing hydrogen peroxide (H 2 O 2 ) in 1.000 Patient
can be stored at 2-30°C for up to 60 days. r
reaction results between the enzyme conjugate and the free acetate buffer. Store at 2-8°C. b 0.500
thyroxine for a limited number of antibody combining sites 2. Working Substrate Solution a
n 0.000
immobilized on the well. G. Stop Solution – 8 ml/vial - Icon
STOP
Pour the contents of the plastic vial labeled Solution ‘A’ into c
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5

One (1) bottle containing a strong acid (1N HCl). Store at the clear vial labeled Solution ‘B’. Place the yellow cap on the e

After the completion of the required incubation period, the 2-8°C. clear vial for easy identification. Mix and label accordingly. fT4l Values in ng/dl

antibody bound enzyme-thyroxine conjugate is separated from H. Product Instructions. Store at 2 - 8°C.
the unbound enzyme-thyroxine conjugate via a wash step. The Note1 : Do not use the working substrate if it looks blue.
activity of the enzyme present on the surface of the well is Note 1: Do not use reagents beyond the kit expiration date. Note 2: Do not use reagents that are contaminated or have
quantitated by reaction with a suitable substrate to produce color. Note 2: Opened reagents are stable for sixty (60) days when bacteria growth.
stored at 2-8°C. Opened reagents are stable for sixty (60) 9.0 TEST PROCEDURE EXAMPLE 1
The employment of several serum references of known free days when stored at 2-8°C. Kit and component stability Mean
thyroxine concentration permits construction of a graph of activity are identified on the label. Before proceeding with the assay, bring all reagents, serum Sample Well Abs Value*
Abs
and concentration. From comparison to the dose response curve, Note 3: Above reagents are for a 96-well microplate. For other kit references and controls to room temperature (20-27°C). I.D. Number (A) (ng/dl)
(B)
an unknown specimen's activity can be correlated with free configurations, please refer to the table at the end of this IFU. **Test Procedure should be performed by a skilled individual
thyroxine concentration. or trained professional** A1 2.658
4.1 Materials Required But Not Provided: Cal A 2.612 0.00
1. Format the microplate wells for each serum reference, control B1 2.566
1. Pipette capable of delivering 50µl & 100µl volumes with a C1 1.919
and patient specimen to be assayed in duplicate. Replace
precision of better than 1.5%. Cal B 1.900 0.45
any unused microwell strips back into the aluminum bag, D1 1.880
2. Dispenser(s) for repetitive deliveries of 0.100ml and 0.350ml
seal and store at 2-8°C E1 1.339
volumes with a precision of better than 1.5%. Cal C 1.306 1.10
F1 1.273
 G1 0.769 dilute the sample. TBG variations in different matrices will In order to validate the inter-assay precision of fT4 AccuBind® 8. Midgeley John, “Direct and Indirect Free Thyroxine Assay Methods
Cal D 0.790 2.00 not allow Free T4 hormone to dilute serially. ELISA test system, one duplicate of each of three pooled sera in Theory and Practice”, Clin Chem, 47, 1353-1363 (2001).
H1 0.811
7. Serum free-thyroxine concentration is dependent upon a (low medium and high ranges of the dose response curve) was 9. Bayer MF and McDougall IR, “Radioimmunoassay of free thyroxine
A2 0.396
Cal E 0.400 5.00 multiplicity of factors: thyroid gland function and its regulation, assayed in 10 assays done over a period of six months that in serum: comparison with clinical findings and results of
B2 0.404 conventional thyroid-function tests”, Clin Chem, 26, 1186-1192
C2 0.215 Thyroxine binding globulin (TBG) concentration, and the involved five different sets of reagents and three different
Cal F 0.217 7.40 binding of Thyroxine to TBG (3, 4). Thus, free-Thyroxine technicians. An inter-assay precision of 6.01 to 10.81% was (1980).
D2 0.219 10. Anthony GW, Jackson RA etal, “Misleading results from
concentration alone is not sufficient to assess the clinical obtained.
E2 1.827 immunoassays of serum free thyroxine in the presence of
Ctrl 1 1.835 0.50 status. TABLE 3 rheumatoid factor”, Clin Chem, 43, 957-962 (1997).
F2 1.843 8. Serum free-thyroxine values may be elevated under conditions Inter-Assay Precision (in ng/dl) 11. Wosilait WD, “A theoretical analysis of the distribution of thyroxine
G2 0.541 such as pregnancy or administration of oral contraceptives.
Ctrl 2 0.557 2.70 Sample N X σ C.V. among sites on the thyroxine binding globulin, thyroid binding
H2 0.573 9. A decrease in free thyroxine values is found with prealbumin and serum albumin”, Res Comm Chem Pathology-
A3 0.951 protein-wasting diseases, certain liver diseases and Low 10 0.480 0.052 10.81% Pharmacology, 16, 541-548 (1977).
Patient 0.964 1.65 administration of testosterone, diphenylhydantoin or Medium 10 1.410 0.085 6.01%
B3 0.976 Revision: 6 Date: 2022-MAY-01 DCO: 1557
salicylates. A table of interfering drugs and conditions, which High 10 3.490 0.279 7.90%
Note 1: Computer data reduction software designed for ELISA Cat #: 1225-300
affect free Thyroxine values, has been compiled by the
assays may also be used for the data reduction. If such 14.2 Sensitivity
Journal of the American Association of Clinical Chemists. Size 96(A) 192(B) 480(D) 960(E)
software is utilized, the validation of the software should be The Free T4 AccuBind® ELISA test system has a sensitivity of
10. The interpretation of Free T4 is complicated by a variety of A) 1ml set 1ml set 2ml set 2ml set x2
ascertained. 0.162 ng/dl. The sensitivity was ascertained by determining the
drugs that can affect the binding of T4 to the thyroid hormone B) 1 (13ml) 2 (13ml) 1(60ml) 2 (60ml)
variability of the 0 ng/dl serum calibrator and using the 2σ (95%

Reagent (fill)
11.0 Q.C. PARAMETERS carrier proteins or interfere in its metabolism to T3. In severe
certainty) statistics to calculate the minimum dose. C) 1 plate 2 plates 5 plates 10 plates
In order for the assay results to be considered valid the non-thyroidal illness (NTI) the assessment of thyroid becomes
following criteria should be met: 14.3 Accuracy D) 1 (20ml) 1 (20ml) 1 (60ml) 2 (60ml)
especially difficult. Since the patients in this category may
1. The absorbance (OD) of calibrator 0 ng/dl should be > 1.3. The Free T4 AccuBind® ELISA test system was compared with a E) 1 (7ml) 2 (7ml) 1 (30ml) 2 (30ml)
suffer from concomitant primary hypothyroidism or from
2. Four out of six quality control pools should be within the compensatory secondary hypothyroidism. In cases like these coated tube radioimmunoassay (RIA) method. Biological F) 1 (7ml) 2 (7ml) 1 (30ml) 2 (30ml)
established ranges. a sensitive TSH evaluation of the patient may be specimens from hypothyroid, euthyroid and hyperthyroid G) 1 (8ml) 2 (8ml) 1 (30ml) 2 (30ml)
12.0 RISK ANALYSIS recommended. Please see Monobind Cat# 325-300. populations were used (The values ranged from 0.1ng/dl –
The MSDS and Risk Analysis Form for this product is available on 11. In rare conditions associated with extreme variations in 8ng/dl). The total number of such specimens was 197. The least
request from Monobind Inc. albumin binding capacity for T4- such as familial square regression equation and the correlation coefficient were
12.1 Assay Performance dysalbuminemic hyperthyroxinemia (FDH) – direct assessment computed for this Free T4 AccuBind® ELISA method in
1. It is important that the time of reaction in each well is held of Free T4 may be misleading. comparison with the predicate method (Table 4).
constant to achieve reproducible results. 12. Circulating antibodies to T4 and hormone binding inhibitors TABLE 4
2. Pipetting of samples should not extend beyond ten (10) may interfere in the performance of the assay. Linear Regression Analysis
minutes to avoid assay drift. 13. Heparin is reported to have in vivo and in vitro effects on free Mean Correlation
3. Highly lipemic, hemolyzed or grossly contaminated T4 levels. Samples from patients undergoing heparin therapy Method (x) Equation Coefficient
specimen(s) should not be used. should be collected well before the administration of the Monobind 1.56 y = 0.1034 + 0.9525x 0.920
4. If more than one (1) plate is used, it is recommended to repeat anticoagulant. EIA “X”
the dose response curve. "NOT INTENDED FOR NEWBORN SCREENING" Predicate 1.59
5. The addition of substrate solution initiates a kinetic reaction, 13.0 EXPECTED RANGES OF VALUES RIA “Y”
which is terminated by the addition of the stop solution. A study of euthyroid adult population was undertaken to
Therefore, the substrate and stop solution should be added in determine expected values for the Free T4 AccuBind® ELISA test Only slight amounts of bias between this method and the
the same sequence to eliminate any time-deviation during system. The mean (X) values, standard deviations (σ) and reference method are indicated by the closeness of the mean
reaction. expected ranges (±2σ) are presented in Table 1. values.
6. Plate readers measure vertically. Do not touch the bottom of TABLE 1 14.4 Specificity:
the wells. The cross-reactivity of the thyroxine antibody used for Free T4
Expected Values for Free T4 ELISA Test System (in ng/dl)
7. Failure to remove adhering solution adequately in the AccuBind® ELISA to selected substances was evaluated by
Adult Pregnancy
aspiration or decantation wash step(s) may result in poor adding massive amounts of the interfering substance to a serum
replication and spurious results. Number of Specimens 89 31 matrix. The cross-reactivity was calculated by deriving a ratio
8. Use components from the same lot. No intermixing of reagents Mean (X) 1.40 1.50 between doses of interfering substance to dose of thyroxine
from different batches. Standard Deviation (σ) 0.30 0.37 needed to displace the same amount of the conjugate.
9. Accurate and precise pipetting, as well as following the exact Expected Ranges (±2 σ) 0.8 – 2.0 0.76 – 2.24
Substance Cross Concentratio
time and temperature requirements prescribed are essential.
It is important to keep in mind that establishment of a range of Reactivity n
Any deviation from Monobind’s IFU may yield inaccurate
values which can be expected to be found by a given method for l–Thyroxine 1.0000 ----
results.
a population of "normal"-persons is dependent upon a multiplicity d-Thyroxine 0.9800 10µg/dl
10. All applicable national standards, regulations and laws,
of factors: the specificity of the method, the population tested and d-Triiodothyronine 0.0150 100µg/dl
including, but not limited to, good laboratory procedures, must
the precision of the method in the hands of the analyst. For these l–Triiodothyronine 0.0300 100µg/dl
be strictly followed to ensure compliance and proper device
reasons each laboratory should depend upon the range of lodothyrosine 0.0001 100µg/ml
usage.
expected values established by the manufacturer only until an Diiodotyrosine 0.0001 100µg/ml
11. It is important to calibrate all the equipment e.g. Pipettes,
in-house range can be determined by the analysts using the Diiodothyronine 0.0001 100µg/ml
Readers, Washers and/or the automated instruments used
method with a population indigenous to the area in which the TBG N/D 40 µg/ml
with this device, and to perform routine preventative
laboratory is located. Albumin N/D 40 mg/ml
maintenance.
14.0 PERFORMANCE CHARACTERISTICS
12. Risk Analysis- as required by CE Mark IVD Directive 98/79/EC Phenylbutazone N/D 10 µg/ml
14.1 Precision
- for this and other devices, made by Monobind, can be Phenytoin N/D 40 µg/ml
The inter and intra assay precisions of the Free T4 AccuBind®
requested via email from [email protected]. Salicylates N/D 500 µg/ml
ELISA test system were determined by analyses on three different
12.2 Interpretation
levels of pooled patient sera. The number (n), mean values (x),
1. Measurements and interpretation of results must be 15.0 REFERENCES
standard deviation (σ) and coefficient of variation (C.V.) for each 1. Barker SB, "Determination of Protein Bound Iodine, Journal
performed by a skilled individual or trained professional.
of these control sera are presented in Table 2 and Table 3. Biological Chemistry, 173, 175 (1948).
2. Laboratory results alone are only one aspect for determining
patient care and should not be the sole basis for therapy, 2. Chopra IJ, Solomon DH, and Ho RS, "A Radioimmunoassay of
In order to validate the intra-assay precision of the Free T4 Thyroxine", J Clinical Endocrinol, 33, 865 (1971).
particularly if the results conflict with other determinants. AccuBind® ELISA test system, twenty replicates of each of three
3. For valid test results, adequate controls and other parameters 3. Young DS, Pestaner L, and Gilberman U, "Effects of Drugs on
pooled sera (low medium and high ranges of the dose response Clinical Laboratory Tests", Clinical Chemistry, 21, 3660 (1975).
must be within the listed ranges and assay requirements. curve) were assayed in the same assay. An intra-assay precision 4. Sterling L, “Diagnosis and Treatment of Thyroid Disease”, CRC
4. If test kits are altered, such as by mixing parts of different kits, of 3.25 to 10.98% was obtained. Press, 19-51 (1975).
which could produce false test results, or if results are 5. Halpern EP and Bordens RW, “Microencapsulated antibodies in
incorrectly interpreted, Monobind shall have no liability. TABLE 2
radioimmunoassay: Determination of free Thyroxine”, Clinical
5. If computer controlled data reduction is used to interpret the Intra-Assay Precision (in ng/dl)
Chemistry, 25, 1561-1563 (1979).
results of the test, it is imperative that the predicted values for Sample N X σ C.V. 6. Stjernholm MR, Alsever RN and Rudolph MC, “Thyroid function
the calibrators fall within 10% of the assigned concentrations. Low 20 0.550 0.061 10.98% tests in diphenylhydantoin-treated patients”, Clin Chem, 21, 1388
6. If a patient, for some reason, reads higher than the highest Medium 20 1.740 0.074 4.26% (1977).
calibrator report as such (e.g. > 7.4 ng/dl). Do not try to High 20 3.250 0.106 3.25% 7. Nelson J.C. and Wilcox, RB. “Analytical performance of Free and
Total thyroxine assays”. Clin. Chem. Vol. 42, 146-154 (1996).