Instructions for use

T3 ELISA 2nd Generation

TF E-2300R

T3 ELISA 2nd Generation
INTRODUCTION
Intended Use
The T3 ELISA is an enzyme immunoassay for the quantitative measurement of total T3 (triiodothyronine) inserum and plasma
PRINCIPLE OF THE TESTThe T3 ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA), based on the principle ofcompetitive binding.
The microtiter wells are coated with a polyclonal goat-anti-mouse antibody. Standards, controls and serum
incubate together with Assay Reagent containing monoclonal anti-T3 antibodies. In the following incubation
with Conjugate the endogenous T3 of a sample competes with the T3-horseradish peroxidase conjugate for alimited number of insolubilized binding sites.
After incubation the unbound conjugate is washed off.
The amount of bound peroxidase conjugate is inversely proportional to the concentration of T3 in the sample.
After addition of the substrate solution, the intensity of colour developed is inversely proportional to theconcentration of T3 in the sample.
WARNINGS AND PRECAUTIONS
1.
2.

This kit is for research use only.
All reagents of this test kit which contain human serum or plasma have been tested and confirmed
negative for HIV I/II, HBsAg and HCV by FDA approved procedures. All reagents, however, should betreated as potential biohazards in use and for disposal.
3. Before starting the assay, read the instructions completely and carefully. Use the valid version ofinstructions for use provided with the kit. Be sure that everything is understood.
4. The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil
pouch and used in the frame provided.
5. Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for eachstep.
6. Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using areservoir for dispensing a substrate solution that had previously been used for the conjugate solution may
turn solution colored. Do not pour reagents back into vials as reagent contamination may occur.
7. Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells.
8. Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
9. Allow the reagents to reach room temperature (21 °C - 26 °C) before starting the test. Temperature willaffect the absorbance readings of the assay. However, values for the samples will not be affected.
10. Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
11. Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
12. Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagentsor specimens may give false results.
13. Handling should be done in accordance with the procedures defined by an appropriate national biohazard
safety guideline or regulation.
14. Do not use reagents beyond expiry date as shown on the kit labels.
15. All indicated volumes have to be performed according to the protocol. Optimal test results are only
obtained when using calibrated pipettes and microtiterplate readers.
16. Do not mix or use components from kits with different lot numbers. It is advised not to exchange wells ofdifferent plates even of the same lot. The kits may have been shipped or stored under different conditions
and the binding characteristics of the plates may result slightly different.
17. Avoid contact with Stop Solution containing 0.5 M H2SO4. It may cause skin irritation and burns.
18. Some reagents contain Proclin 300, BND and/or MIT as preservatives. In case of contact with eyes or skin,flush immediately with water.
19. TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with anabundant volume of water and skin with soap and abundant water. Wash contaminated objects before
reusing them. If inhaled, take the person to open air.
20. Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national
biohazard safety guideline or regulation.
21. For information on hazardous substances included in the kit please refer to Material Safety Data Sheets.
Safety Data Sheets for this product are available upon request directly from the manufacturer.

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REAGENTS
Reagents provided
TF E-2331 Microtiterwells
12 x 8 (break apart) strips, 96 wells;
Wells coated with goat anti-mouse antibody (polyclonal).
Standards
ready to use
Cat. no.
TF E-2301

Standard
Standard A (0)Concentration
0 ng/ml

Volume/Vial
0.75 ml

TF E-2302

Standard B (1)0.5 ng/ml

0.75 ml

TF E-2303

Standard C (2)1.0 ng/ml

0.75 ml

TF E-2304

Standard D (3)2.5 ng/ml

0.75 ml

TF E-2305

Standard E (4)5.0 ng/ml

0.75 ml

TF E-2306
Contain non-mercury preservative.

Standard F (5)10.0 ng/ml

0.75 ml

TF E-2351 Control Low
1 vial, 0.75 mL, ready to use;
For control values and ranges please refer to vial label or QC-Datasheet.
Contain non-mercury preservative.
TF E-2352 Control High
1 vial, 0.75 mL, ready to use;
For control values and ranges please refer to vial label or QC-Datasheet.
Contain non-mercury preservative.
TF E-2313 Assay Reagent
1 vial, 6 mL, ready to use,
Contains buffer, binding protein inhibitors and anti-T3 mAb.
Contains non-mercury preservative.
TF E-2340 Enzyme Conjugate
1 vial, 6 mL, ready to use,
T3 conjugated to horseradish peroxidase;
Contains non-mercury preservative.
TF E-0055 Substrate Solution
1 vial, 12 mL, ready to use,
Tetramethylbenzidine (TMB).
Hazards
identification:
H360D May damage the unborn child.
FR E-0080 Stop Solution
1 vial, 14 mL, ready to use,
contains 0.5 M H2SO4,
Avoid contact with the stop solution. It may cause skin irritations and burns.
Hazards
identification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.
FR E-0030 Wash Solution
1 vial, 30 mL (40X concentrated),
See “Reagent Preparation“.
Note: Additional Standard A for sample dilution is available upon request.
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Materials required but not provided

A microtiter plate calibrated reader (450 ± 10 nm).
Calibrated variable precision micropipettes.
Absorbent paper.
Distilled or deionized waterTimer
Semi logarithmic graph paper or software for data reduction

Storage Conditions
When stored at 2 °C to 8 °C unopened reagents will retain reactivity until expiration date. Do not use reagents
beyond this date.
Opened reagents must be stored at 2 °C to 8 °C. Microtiter wells must be stored at 2 °C to 8 °C. Once the foil
bag has been opened, care should be taken to close it tightly again.
Opened kits retain activity for 8 weeks if stored as described above.
Reagent Preparation
Bring all reagents and required number of strips to room temperature prior to use.
Wash Solution
Add deionized water to the 40X concentrated Wash Solution.
Dilute 30 mL of concentrated Wash Solution with 1170 mL deionized water to a final volume of 1200 mL.
The diluted Wash Solution is stable for 2 weeks at room temperature.
Disposal of the Kit
The disposal of the kit must be made according to the national regulations. Special information for this productis given in the Material Safety Data Sheet.
Damaged Test Kits
In case of any severe damage to the test kit or components, the manufacturer has to be informed in writing, atthe latest, one week after receiving the kit. Severely damaged single components should not be used for a testrun. They have to be stored until a final solution has been found. After this, they should be disposed according
to the official regulations.
SPECIMEN COLLECTION AND PREPARATION
Serum or plasma (EDTA- or heparin plasma) can be used in this assay.
Do not use haemolytic, icteric or lipaemic specimens.
Please note: Samples containing sodium azide should not be used in the assay.
Specimen Collection
Serum:
Collect blood by venipuncture (e.g. Sarstedt Monovette for serum), allow to clot, and separate serum bycentrifugation at room temperature. Do not centrifuge before complete clotting has occurred.
Plasma:Whole blood should be collected into centrifuge tubes containing anti-coagulant (e.g. Sarstedt Monovette withthe appropriate plasma preparation) and centrifuged immediately after collection.
Specimen Storage and Preparation
Specimens should be capped and may be stored for up to 48 hours at 2 °C to 8 °C prior to assaying.
Specimens held for a longer time (up to 30 days) should be frozen only once at -20 °C prior to assay. Thawed
samples should be inverted several times prior to testing.
Specimen Dilution
If in an initial assay, a specimen is found to contain more than the highest standard, the specimens can be
diluted with Standard A and reassayed as described in Assay Procedure.
For the calculation of the concentrations this dilution factor has to be taken into account.
Example:a) dilution 1:2:

30 µL sample + 30 µL Standard A (mix thoroughly)

ASSAY PROCEDURE
General Remarks
 All reagents and specimens must be allowed to come to room temperature before use. All reagents must be
mixed without foaming.
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



Once the test has been started, all steps should be completed without interruption.
Use new disposal plastic pipette tips for each standard, control or sample in order to avoid crosscontamination.
Absorbance is a function of the incubation time and temperature. Before starting the assay, it isrecommended that all reagents are ready, caps removed, all needed wells secured in holder, etc. This will
ensure equal elapsed time for each pipetting step without interruption.
As a general rule the enzymatic reaction is linearly proportional to time and temperature.

Test Procedure
Each run must include a standard curve.
1.

Secure the desired number of Microtiter wells in the frame holder.

2.

Dispense 50 µL of each Standard, control and sample with new disposable tips into appropriatewells.
It is important to add first the standards or samples before adding the Assay Reagent.

3.

Add 50 µL of Assay Reagent to each well.
Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.

4.

Incubate for 30 minutes at room temperature (20 °C – 27 °C).

5.

Dispense 50 µL Enzyme Conjugate into each well.
Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.

6.

Incubate for 30 minutes at room temperature (20 °C – 27 °C).

7.

Briskly shake out the contents of the wells.
Rinse the wells 5 times with diluted Wash Solution (300 µL per well). Strike the wells sharply on
absorbent paper to remove residual droplets.
Important note:
The sensitivity and precision of this assay is markedly influenced by the correct performance of thewashing procedure!

8.

Add 100 µL of Substrate Solution to each well.

9.

Incubate for 10 minutes at room temperature (20 °C – 27 °C).

10. Stop the enzymatic reaction by adding 100 µL of Stop Solution to each well.
11. Determine the absorbance (OD) of each well at 450 ± 10 nm with a microtiter plate reader.
It is recommended that the wells be read within 10 minutes after adding the Stop Solution.
Calculation of Results
1. Calculate the average absorbance values for each set of standards, controls and samples.
2. Using semi-logarithmic graph paper, construct a standard curve by plotting the mean absorbance obtained
from each standard against its concentration with absorbance value on the vertical (Y) axis andconcentration on the horizontal (X) axis.
3. Using the mean absorbance value for each sample determine the corresponding concentration from thestandard curve.
4. Automated method: The results in the Instructions for Use have been calculated automatically using a
4 Parameter curve fit. (4 Parameter Rodbard or 4 Parameter Marquardt are the preferred methods). Other
data reduction functions may give slightly different results.
5. The concentration of the samples can be read directly from this standard curve. Samples with concentrations
higher than that of the highest standard have to be further diluted or reported as > 10 ng/mL. For thecalculation of the concentrations this dilution factor has to be taken into account.
Example of Typical Standard Curve
The following data is for demonstration only and cannot be used in place of data generations at the time ofassay.
Standard
Optical Units (450 nm)
Standard A (0.0 ng/mL)

1.89

Standard B (0.5 ng/mL)

1.42

Standard C (1.0 ng/mL)

1.13

Standard D (2.5 ng/mL)

0.58

Standard E (5.0 ng/mL)

0.36Standard F (10.0 ng/mL)

0.20

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EXPECTED NORMAL VALUES
It is strongly recommended that each laboratory should determine its own normal and abnormal values.
In a study conducted with euthyroid adult population using the Total T3 ELISA the following values areobserved:Population

ValidN

Range(ng/mL)

Mean(ng/mL)

2.5th - 97.5th
Percentile (ng/mL)

Median(ng/mL)

Males andfemales

140

0.63 - 1.99

1.19

0.75 - 1.70

1.18

QUALITY CONTROLGood laboratory practice requires that controls be run with each standard curve. A statistically significant
number of controls should be assayed to establish mean values and acceptable ranges to assure proper
performance.
It is recommended to use control samples according to state and federal regulations. The use of control
samples is advised to assure the day to day validity of results. Use controls at both normal and pathologicallevels.
The controls and the corresponding results of the QC-Laboratory are stated in the QC certificate added to thekit. The values and ranges stated on the QC sheet always refer to the current kit lot and should be used fordirect comparison of the results.
It is also recommended to make use of national or international Quality Assessment programs in order toensure the accuracy of the results.
Employ appropriate statistical methods for analysing control values and trends. If the results of the assay do
not fit to the established acceptable ranges of control materials results should be considered invalid.
In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration
dates of reagents, storage and incubation conditions, aspiration and washing methods.
After checking the above mentioned items without finding any error contact your distributor or themanufacturer directly.
PERFORMANCE CHARACTERISTICS
Assay Dynamic Range
The range of the assay is between 0.1 – 10 ng/mL.
Specificity of Antibodies (Cross Reactivity)
The following substances were tested for cross reactivity of the assay:
Substance
Cross Reactivity (%)
I-Triiodothyronine
100
I-Thyroxine
0.37
Reverse T3
0.75
D-Thyroxine
0.1
3,5-Diiodo-L-Thyrosine
0.2
4-Phenoxyphenol
0.2
Sensitivity
The analytical sensitivity of the T3 ELISA was calculated by subtracting 2 standard deviations from the mean of
20 replicate analyses of the Standard A (S0) and was found to be < 0.1 ng/mL.

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Reproducibility
Intra Assay
The within assay variability is shown below:
SamplenMean (ng/mL)
CV (%)
120

1.15

6.61

220

1.69

6.54

320

2.85

3.59

Inter Assay
The between assay variability is shown below:
SamplenMean (ng/mL)
CV (%)
110

0.94

6.37

210

1.52

5.23

310

2.86

6.73

Recovery
Samples have been spiked by adding T3 solutions with known concentrations in a 1:1 ratio.
The % recovery has been calculated by multiplication of the ratio of the measurements and the expected values
with 100 (expected value = (endogenous T3 + added T3) / 2; because of a 1:2 dilution of serum with spikematerial).
Sample 1

Sample 2

Sample 3

Concentration [ng/mL]

1.09

1.38

2.37

Average Recovery [%]

102.5

103.0

105.3

from99.8

88.4

98.9

to106.9

109.2

108.7

Sample 1

Sample 2

Sample 3

Concentration [ng/mL]

3.70

3.90

3.36

Average Recovery [%]104.8100.1100.1

from

100.0

94.9

95.1

to111.0

106.8

107.3

Range of Recovery [%]Linearity

Range of Recovery [%]LIMITATIONS OF USE
Reliable and reproducible results will be obtained when the assay procedure is performed with a complete
understanding of the package insert instruction and with adherence to good laboratory practice.
Any improper handling of samples or modification of this test might influence the results.
Drug Interferences
A table of interfering drugs and conditions which affect total Triiodothyronine values has been compiled by theJournal of the American Association of Clinical Chemists (3)High-Dose-Hook EffectNo hook effect was observed in this test.
REFERENCES / LITERATURE
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 Triiodothyronine,"
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. 19 51 (1975).

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Symbols:
Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContentCaution

Catalogue
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Effective: 2018-06-21

Contains sufficient for<n> tests

For research use
only!
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