Package Insert

Free T3 (FT3) ELISA

Assay kit

Catalog Number:
FT331-K01
1 x 96 well ELISA kit
For Research Use Only
v. 1.0

Eagle Biosciences, Inc.

20A NW Blvd, Suite 112, Nashua, NH 03063
Phone: 617-419-2019 Fax: 617-419-1110
www.EagleBio.com
Intended Use:
Eagle Biosciences Free T3 (Triiodothyronine) ELISA Assay kit is intended for the quantitative
determination of Free T3 (Triiodothyronine) in serum by enzyme linked immunoassay (ELISA).
The Free T3 ELISA Assay kit is for research use only and not to be used in diagnostic procedures.

Circulating thyroid hormones (thyroxine, T4 and triiodothyronine, T3) are distributed into two
compartments; a major protein bound one, and a minor free fraction (0.03 % of T4 and 0.3 % of
T3). Variations in total thyroid hormone in blood may result from either changes of binding
protein concentrations, or thyroid hormone synthesis. Thyroid disorders exist only if a net
change of free unbound fractions occurs persistently. Therefore the true determination of
thyroid status will be the measurement of the free hormones.

Among the thyroid hormones produced in the thyroid gland triiodothyronine (3,5,3’-triiodo-L-
thyronine, T3) is regarded as the most biologically active molecule. It is produced up to 80 % by
the de-iodination of tetraiodothyronine (T4) in peripheral tissues. Hyperthyroidism is generally
associated with an increase of FT3 concentration, and in several cases a single increased FT3
indicates a T3-thyrotoxicosis. In the follow-up of patients who are subject to a therapy with
triiodothyronine the FT3 measurement is also necessary.

The present 1-step free T3 radioimmunoassay enables a reliable, clinically validated

determination of FT3 in the concentration range from 0 to 40 pmol/l (0 - 26 pg/ml).

Principle of the Test:

The Free T3 ELISA Assay Kit is a competitive enzyme immunoassay with a step by step
incubation.

During the first incubation triiodothyronine of the patient samples and calibrators bind to the
monoclonal anti-triiodothyronine antibodies immobilized on the solid phase of the microtiter
plate. Following an incubation period of 45 min, unbound components are separated from the
solid-phase immune complexes by a wash step.

In a second incubation step of 15 min the triiodothyronine labeled to horseradish peroxidase

(HRP) binds to the free antibodies on the solid phase. The more triiodothyronine is present in
the sample, the less HRP-labeled triiodothyronine is bound on the solid phase.

Horseradish peroxidase converts the colorless substrate solution of 3,3’,5,5’-

tetramethylbenzidine (TMB) added into a blue product. This enzyme reaction is stopped by
dispensing an acidic solution (HCl) into the wells after 20 min at room temperature turning the
solution from blue to yellow.

The optical density (OD) of the solution at 450 nm is indirectly proportional to the amount of
triiodothyronine. The standard curve is established by plotting the concentrations of the
standards (x-axis) and their corresponding OD values (y-axis) measured. The concentration of
2

the specimen is directly read off the standard curve.

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Free T3 (FT3) ELISA Assay Kit
Catalog Number FT331-K01
Samples:
 Blood is taken by venipuncture. After clotting, the serum is separated by centrifugation.
Do not use plasma, hemolysed or lipemic sera.

 The samples may be kept at 2 - 8 °C for up to 2 days. Long-term storage requires -20 °C.
Repeated freezing and thawing should be avoided. If required, the samples have to be
initially frozen in aliquots.

 Prior to assay, allow the samples to reach room temperature. Take care to agitate serum
samples gently in order to ensure homogeneity.

Note: Before assayed the sera have to be free of any particulate matter (centrifuge, if
necessary, and use the clear supernatants only).

Materials required:

 micropipette 100 - 1000 µl

 micropipette 10 - 100 µl
 multi-channel pipette 50 - 200 µl
 trough for multi-channel pipette
 8-channel wash comb with vacuum pump and waste bottle or microplate washer
 microplate reader with optical filters for 450 nm and 620 nm or 690 nm
 horizontal shaker
 graduated cylinders
 distilled water
 foil to seal microtiterplate

Size and Storage:

 The Free T3 ELISA Assay Kit has been designed for 96 determinations. This is sufficient for
the analysis of 41 unknown samples as well as for calibrators and controls assayed in
duplicates.

 The expiry date of each component is reported on its respective label that of the complete
FT3 ELISA Assay kit on the box labels.

 Upon receipt, all components of the FT3 ELISA Assay Kit have to be kept at 2 - 8 °C,
preferably in the original Free T3 ELISA Assay kit box.
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Free T3 (FT3) ELISA Assay Kit
Catalog Number FT331-K01
Preparation before use:
Please, handle the following components carefully:

A Allow the sealed microplate to reach room temperature before opening. Unused wells
should be stored refrigerated and protected from moisture in the original bag. Carefully
resealed it can be used for 8 weeks.

B Prepare a sufficient amount of washing solution by diluting the concentrated wash buffer (B)
1 + 19 with distilled or de-ionized water. Dilute 1 bottle (14 ml) of the concentrate with
266ml of distilled water. B should be free of crystals before dilution, otherwise dissolve by
warming up to max. 37 °C. The diluted washing solution can be stored at 2 - 8 °C up to 30
days.

E Avoid exposure of substrate solution (E) to light.

Assay Procedure:
 After opening all Free T3 ELISA Assay kit components are stable for at least 2 months,
provided proper storage at 2 - 8 °C.
 Bring all reagents to room temperature (20…25°C) before use.
 Duplicates are recommended.

1. Pipette into all wells according to assay scheme

- 80 µl start buffer.

2. Pipette into corresponding wells

- 20 µl calibrators
- 20 µl control
- 20 µl of the respective patient samples.

3. Cover the plate and incubate for 45 min at 37 °C while shaking (600-800 rpm).

4. Aspirate or “flick out” by striking the wells sharply onto absorbent paper to remove any
residual droplets. Wash 4 times with 300 µl washing solution (diluted from B) with 5
seconds soaking time each.

5. Add 100 µl of conjugate (D) to each well.

6. Cover the plate and incubate for 15 min at 37 °C while shaking (600-800 rpm).
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Free T3 (FT3) ELISA Assay Kit
Catalog Number FT331-K01
7. Aspirate or “flick out” by striking the wells sharply onto absorbent paper to remove any
residual droplets. Wash 4 times with 300 µl washing solution (diluted from B) with 5
seconds soaking time each.

8. Add 100 µl substrate solution (E) to each well, shake shortly.

9. Incubate for 20 min in the dark at RT.

10. Add 100 µl stop solution (F) to each well, shake well for 1 - 2 min..

11. Read the optical density (OD) at 450 nm versus 620 or 690 nm within 30 min after
adding the stop solution.

Data Processing:
 The standard curve is established by plotting the mean OD-values of the calibrators 0 - 5
on the ordinate, y-axis, versus their respective FT3 concentrations on the abscissa, x-axis
(log scale).
 The FT3 concentrations of the control and the unknown samples are directly read off in
pmol/l from the measured OD values.
 The Free T3 ELISA Assay Kit may be used also with computer assisted analysis software
integrated in photometers. We recommend 4 parameter fit.

Typical Example:
L
Do not use for evaluation!

Sample OD (a) OD (b) OD pmol/l

450 nm 450 nm (me
an)

Calibrator 0 2.284 2.135 2.210 0

Calibrator 1 1.991 1.847 1.919 1
Calibrator 2 1.703 1.731 1.717 2
Calibrator 3 1.062 1.022 1.042 11.7
Calibrator 4 0.729 0.694 0.712 30.6
Calibrator 5 0.493 0.459 0.476 63

Sample 1 1.097 1.120 1,109 9.5

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Free T3 (FT3) ELISA Assay Kit
Catalog Number FT331-K01
Standard Curve:

L
Typical example

Criteria of validation:
Specimens with concentrations higher than calibrator 5 should be diluted further by the start
buffer (H) and the FT3 concentration should be calculated by the applied dilution factor. Any
extrapolation of the standard curve to Free T3 concentration above the nominal value of
calibrator 5 is not permitted.

Reference Values:

Range of free T3 concentrations determined with the Free T3 ELISA Assay kit in serum samples
collected between 9 and 11 a.m. from 200 apparently healthy people (both males and females)
at the age of 18 - 45 was 2,5-7,5 pmol/l. These limits should be considered as guidelines only.

It is highly recommended for each laboratory to determine its own reference range of free T3
concentrations.

Conversion factors

pmol/l x 0.65 = pg/ml

6

pg/ml x 1.54 = pmol/l

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Free T3 (FT3) ELISA Assay Kit
Catalog Number FT331-K01
Characteristic Assay Data:
Specificity:

Cross-reaction of monoclonal T3 antibodies used in the Free T3 ELISA Assay kit with different
thyroids is shown in the table below:

Thyroid Cross-
reaction
D-triiodothyronin 100 %
D-thyroxin ≤ 0.1 %
L-thyroxin ≤ 0.1 %
r-Triiodothyronine ≤ 0.1 %
Diiodotyrosine ≤ 0.1 %

Analytical sensitivity (lower detection limit):

Analytical sensitivity of Free T3 ELISA assay, or the lowest detectable concentration that can be
distinguished from zero calibrator, is 0.5 pmol/l. It is defined as mean OD of 10 replicates of
Calibrator 0 minus two standard deviations.

Intra- and inter-assay variation

To determine intra-assay CV 8 serum samples were assayed in 9 replicates each. The results are
shown below.

Mean conc.
Sample of free T3, SD CV
pmol/l
HS 1 1.5 0.49 22.5
HS 2 3.8 0.35 9.8
HS 3 5.4 0.24 4.5
HS 4 5.8 0.31 5.4
HS 5 6.2 0.37 6.0
HS 6 7.2 0.25 13.1
HS 7 20.2 0.34 14.2
HS 8 39.3 0.87 12.6 7
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Free T3 (FT3) ELISA Assay Kit
Catalog Number FT331-K01
To determine inter-assay CV 8 serum samples were assayed 3 times by different operators with
1-week interval. Each specimen was assayed in 9 replicates. The results are shown below.

assay 1 assay 2 assay 3

free T3, free T3, free T3,
Sample SD CV
pmol/l pmol/l pmol/l
HS 1 1.75 1.84 1.66 0.232 14.0
HS 2 1.82 1.77 1.32 0.275 16.8
HS 3 3.70 3.54 4.25 0.372 9.7
HS 4 5.62 5.50 5.26 0.183 3.4
HS 5 6.14 6.22 6.00 0.111 1.8
HS 6 8.06 7.8 8.2 0.203 2.5
HS 7 19.81 18.6 18.2 0.838 4.4
HS 8 39.0 38.2 39.4 0.611 1.6

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Free T3 (FT3) ELISA Assay Kit
Catalog Number FT331-K01
 Assay Scheme:

Patient samples
Step Activity Material CAL 0 - 5 C
1, 2 etc.

1 Pipette Start buffer (H) 80 µl 80 µl 80

2 Pipette Calibrators,
Control,

Pipette Samples 20 µl 20 µl 20 µl

3 Incubate Plate (A) 45 min at 37 °C while shaking (600-800 rpm)

Aspirate or decant put sharply onto absorbent tissue

4
Pipette Washing solution 4 x 300 µl 4 x 300 µl 4 x 300 µl
made from B

5 Pipette Conjugate (D) 100 µl 100 µl 100 µl

6 Incubate Plate (A) 15 min at 37 °C while shaking (600-800 rpm)

Aspirate or decant put sharply onto absorbent tissue

7
Pipette Washing solution 4 x 300 µl 4 x 300 µl 4 x 300 µl
made from B

8 Pipette Substrate (E) 100 µl 100 µl 100 µl

9 Incubate Plate (A) 20 min at RT in the dark

10 Pipette and shake for 1-2 Stop solution (F) 100 µl 100 µl 100 µl
min

11 Measure OD at 450 nm versus 620 nm (or 690 nm) within 30 min

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Free T3 (FT3) ELISA Assay Kit
Catalog Number FT331-K01
Safety Precautions:
 This Eagle Biosciences Free T3 (Triiodothyronine) ELISA Assay kit is
for research use only. Follow the working instructions carefully. This
instruction manual is valid only for the present FT3 ELISA Assay kit
with the given composition. An exchange of single components is
not in agreement with CE regulations.

 The expiration dates stated on the respective labels are to be

observed. The same relates to the stability stated for reconstituted
reagents.

 Do not use or mix reagents from different lots.

 Do not use reagents from other manufacturers.

 Avoid time shift during pipetting of reagents.

 All reagents should be kept at 2 - 8 °C before use in the original

shipping container.

 Some of the reagents contain small amounts of Kathon MW as

preservative. They must not be swallowed or allowed to come into
contact with skin or mucosa.

 Source materials derived from human body fluids or organs used

in the preparation of this FreeT3 ELISA Assay kit were tested and
found negative for HBsAg and HIV as well as for HCV antibodies.
However, no known test guarantees the absence of such viral
agents. Therefore, handle all components and all patient samples
as if potentially hazardous.

 Since the Free T3 ELISA Assay kit contains potentially hazardous

materials, the following precautions should be observed:

- Do not smoke, eat or drink while handling Free T3 ELISA Assay

kit material,
- Always use protective gloves,
- Never pipette material by mouth,
- Wipe up spills promptly, washing the affected surface thoroughly with a
decontaminant.
10

 In any case GLP should be applied with all general and individual regulations to
the use of this Free T3 ELISA Assay kit.
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Free T3 (FT3) ELISA Assay Kit
Catalog Number FT331-K01
References:

1. Bartalena L, Bogazzi F, Pinchera A. Thyroid function tests diagnostic protocols for

investigation of thyroid dysfunction. Ann Ist Super Sanita 1991; 27(3):531-9.
2. National Academy of Clinical Biochemistry. International Thyroid Testing Guidelines.
Implications for Clinical Practice. 2001.
3. Reiners Chr. Diagnostische Strategien: Bestimmung von Schilddrüsenhormonen im Serum.
Internist 1988; 29: 529-32.
4. Carayon P et al. International Symposium on Free Thyroid Hormones, Venice. In: Ekins R et
al. (eds.) Excerpta Medica 1979: 181-93.
5. Robbins J, Rall JE. Recent Prog Horm Res 1957; 13: 161-202.
6. Avalos E at al. J Nucl Med 1986; 27: 1702-5.
7. Levinson SS. J Clin Immunoassay 1992; 15: 108-115.

Warranty Information
Eagle Biosciences, Inc. warrants its Product(s) to operate or perform substantially in
conformance with its specifications, as set forth in the accompanying package insert. This
warranty is expressly limited to the refund of the price of any defective Product or the
replacement of any defective Product with new Product. This warranty applies only when the
Buyer gives written notice to the Eagle Biosciences within the expiration period of the Product(s)
by the Buyer. In addition, Eagle Biosciences has no obligation to replace Product(s) as result of a)
Buyer negligence, fault, or misuse, b) improper use, c) improper storage and handling, d)
intentional damage, or e) event of force majeure, acts of God, or accident.

Eagle Biosciences makes no warranties, either expressed or implied, except as provided herein,
including without limitation thereof, warranties as to marketability, merchantability, fitness for a
particular purpose or use, or non-infringement of any intellectual property rights. In no event
shall the company be liable for any indirect, incidental, or consequential damages of any nature,
or losses or expenses resulting from any defective product or the use of any product.
Product(s) may not be resold, modified, or altered for resale without prior written approval from
Eagle Biosciences, Inc.

For further information about the Free T3 (Triiodothyronine) ELISA Assay , its application
or the procedures in this kit insert, please contact the Technical Service Team at Eagle
Biosciences, Inc. at info@eaglebio.com or at 866-411-8023.
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Free T3 (FT3) ELISA Assay Kit
Catalog Number FT331-K01