Instructions for use

Free Estriol ELISAFR E-2100R

Free Estriol ELISA
1. INTRODUCTION
1.1 Intended Use
The Free Estriol ELISA is an enzyme immunoassay for the quantitative measurement of free estriol(unconjugated estriol) in serum during the second half of pregnancy.
2. PRINCIPLE OF THE TESTThe Free Estriol ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA), based on theprinciple of competitive binding.
The microtiter wells are coated with a polyclonal [rabbit] antibody directed towards an antigenic site on theEstriol molecule. Endogenous Estriol of a sample competes with an Estriol-horseradish peroxidase conjugate
for binding to the coated antibody. After incubation the unbound conjugate is washed off.
The amount of bound peroxidase conjugate is inversely proportional to the concentration of Estriol in thesample. After addition of the substrate solution, the intensity of colour developed is inversely proportional tothe concentration of Estriol in the sample.
3. 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 of thepackage insert 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 a reservoir
for dispensing a substrate solution that had previously been used for the conjugate solution may turnsolution coloured. 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 to 26 °C) before starting the test. Temperature willaffect the absorbance readings of the assay. However, values for the donor 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 microtiter plate 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 Safety Data Sheets.
Safety Data Sheets for this product are available upon request directly from the manufacturer.

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4. REAGENTS
4.1 Reagents provided
FR E-2131
Content:

Microtiterwells
12x8 (break apart) strips; 96 wells; Wells coated with anti-Estriol antibody (polyclonal).

Standards and Controls - Ready to use
StandardConcentration

Volume/Vial

FR E-2101

Cat. no.

ComponentStandard A

0 ng/ml

1 ml

FR E-2102

Standard B

0.3 ng/ml

1 ml

FR E-2103

Standard C

1.2 ng/ml

1 ml

FR E-2104

Standard D

4.0 ng/ml

1 ml

FR E-2105

Standard E

15 ng/ml

1 ml

FR E-2106

Standard F

40 ng/ml

1 ml

FR E-2151

Control 1Control 2

For control values andranges please refer to viallabel or QC-Report

1 ml

FR E-2152

1 ml

Contain non-mercury preservatives
FR E-2140
Content:
Volume:Enzyme Conjugate - Ready to use
Estriol conjugated to horseradish peroxidase; contains non-mercury preservative.
1 x 14 ml

ME E-0055
Substrate Solution - Ready to use
Content:
Tetramethylbenzidine (TMB)
Volume:1 x 14 ml
Hazards
identification:
H360D May damage the unborn child.
FR E-0080
Content:
Volume:Hazards
identification:

Stop Solution - Ready to use
Contains 0.5 M H2SO4; avoid contact with the stop solution. It may cause skin irritations
and burns.
1 x 14 ml

H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.
FR E-0030
Volume:Wash Solution - 40x concentrated
1 x 30 ml
see “Preparation of Reagents”.

Note: Additional Standard A for sample dilution is available upon request.
4.2 Materials required but not provided-A microtiter plate calibrated reader (450 nm, with reference wavelength at 620 nm - 630 nm)
Calibrated variable precision micropipettes.
Absorbent paper.
Distilled waterTimer
Semi logarithmic graph paper or software for data reduction

4.3 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 thefoil 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.

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4.4 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.
4.5 Disposal of the Kit
The disposal of the kit must be made according to the national regulations. Special information for thisproduct is given in the Safety Data Sheets.
4.6 Damaged Test Kits
In case of any severe damage to the test kit or components, the manufacturer has to be informed in writing,at the latest, one week after receiving the kit. Severely damaged single components should not be used fora test run. They have to be stored until a final solution has been found. After this, they should be disposed
according to the official regulations.
5. SPECIMEN COLLECTION AND PREPARATION
Serum should be used in this assay.
Note: Samples containing sodium azide should not be used in the assay.
In general it should be avoided to use haemolytic, icteric or lipaemic specimens. For further informationrefer to chapter “Interfering Substances”.
5.1 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. Donors
receiving anticoagulant therapy may require increased clotting time.
5.2 Specimen Storage and Preparation
Specimens should be capped and may be stored for up to 7 days at 2 °C to 8 °C prior to assaying.
Specimens held for a longer time (up to 19 months) should be frozen only once at -20 °C prior to assay.
Thawed samples should be inverted several times prior to testing.
5.3 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 1:10:b) Dilution 1:100:

10 µl Serum + 90 µl Standard A (mix thoroughly)
10 µl dilution a) 1:10 + 90 µl Standard A (mix thoroughly).

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

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6.2 Test Procedure
Each run must include a Standard curve.
1.

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

2.

Dispense 10 µl of each Standard, Control and samples with new disposable tips into appropriate
wells.

3.

Dispense 100 µl Enzyme Conjugate into each well.
Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.

4.

Incubate for 60 minutes at room temperature.

5.

Briskly shake out the contents of the wells.
Rinse the wells 4 times with 400 µl diluted Wash Solution per well, if a plate washer is used - orrinse the wells 4 times with 300 µl diluted Wash Solution per well for manual washing
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!

6.

Add 100 µl of Substrate Solution to each well.

7.

Incubate for 30 minutes at room temperature.

8.

Stop the enzymatic reaction by adding 100 µl of Stop Solution to each well.

9.

Determine the absorbance (OD) of each well at 450 nm (reading) and at 620 - 630 nm(background subtraction, recommended) with a microtiter plate reader.
It is recommended that the wells be read within 10 minutes after adding the Stop Solution.

6.3 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 absorbanceobtained 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 withconcentrations higher than that of the highest standard have to be further diluted or reported as> 40 ng/ml. For the calculation of the concentrations this dilution factor has to be taken into account.
6.3.3 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)

1.79

Standard B (0.3 ng/ml)

1.48

Standard C (1.2 ng/ml)

1.18Standard D (4.0 ng/ml)

0.81Standard E (15.0 ng/ml)

0.52

Standard F (40.0 ng/ml)

0.38

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7. EXPECTED NORMAL VALUES
It is strongly recommended that each laboratory should determine its own normal and abnormal values.
7.1 Normal healthy adults
In a study conducted with apparently normal healthy adults, using the Free Estriol ELISA the following
values are observed:
PopulationnMean[ng/ml]Median[ng/ml]5th - 95th
Percentile[ng/ml]2.5th - 97.5th
Percentile[ng/ml]Range (min - max)
[ng/ml]Males

420.359

0.367

0.146 - 0.573

0.077 - 0.878

0.075 - 0.987

Females(notpregnant)

43

0.348

0.286

0.057 - 0.822

0.038 - 1.027

0.009 - 1.307

7.2 Values during pregnancy(p.m. = post menstruationem)
Expected range

Twin pregnancy[ng/ml]Week ofgestation
p.m.

[ng/ml][ng/ml]120.3 - 1.0

22 - 23

2.7 - 16

3 - 18130.3 - 1.1

24 - 25

2.9 - 173 - 2014

0.4 - 1.6

26 - 27

3.0 - 18

4 - 2115

1.0 - 4.4

28 - 29

3.2 - 20

4 - 2216

1.4 - 6.5

30 - 31

3.6 - 225 - 25

17

1.5 - 6.632 - 33

4.6 - 23

6 - 3918

1.6 - 8.534 - 35

5.1 - 25

7 - 39191.9 - 1136 - 37

7.2 - 29

9 - 3820

2.1 - 1338 - 39

7.8 - 37

13 - 40

21

2.6 - 1440 - 42

8.0 - 39---

Week ofgestation
p.m.

Expected range

8. QUALITY CONTROLGood laboratory practice requires that controls be run with each calibration 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 tothe kit. The values and ranges stated on the QC sheet always refer to the current kit lot and should be used
for direct 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 sample 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.
9. PERFORMANCE CHARACTERISTICS
9.1 Assay Dynamic Range
The range of the assay is between 0.021 ng/ml – 40 ng/ml.

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9.2 Specificity of Antibodies (Cross Reactivity)
The following substances were tested for cross reactivity of the assay:
Added steroid
Estriol (E3)
Testosterone
Estradiol (E2)
Estrone (E1)
Cortisol

Concentrationof steroid
40 ng/ml
16 ng/ml
2 ng/ml
2 ng/ml
800 ng/ml

OD 450
0.39
1.758
1.579
1.712
1.775

Measuredconcentration
39.67 ng/ml
n.d.
n.d.
n.d.
n.d.

n.d. = non detectable
9.3 Sensitivity
The analytical sensitivity of the ELISA was calculated by subtracting 2 standard deviations from the mean of
20 replicate analyses of the Standard A and was found to be 0.021 ng/ml.
9.4 Reproducibility
9.4.1 Intra Assay
The within assay variability is shown below:
SamplenMean [ng/ml]

CV (%)

120

2.1

4.7

220

6.2

3.2

320

14.6

3.0

9.4.2 Inter Assay
The between-assay variability is shown below:
SamplenMean [ng/ml]

CV (%)

1122.1

4.6

212

5.7

8.5

31213.3

9.5

9.5 Recovery
Recovery of the ELISA was determined by adding increasing amounts of the analyte to three different
sample sera containing different amounts of endogenous analyte. Each sample (non-spiked and spiked) was
assayed and analyte concentrations of the samples were calculated from the standard curve. The percentage
recoveries were determined by comparing expected and measured values of the samples.
Sample 1

Sample 2

Sample 3

Concentration [ng/ml]

1.3

3.6

7.8

Average Recovery

100.8

101.8

106.9

from89.0

92.3

98.5

to103.8

109.8112.3

Sample 1

Sample 2

Sample 3

Concentration [ng/ml]

2.6

7.2

15.6

Average Recovery

98.2

99.7

100.2

Range of Recovery [%]9.6 Linearity

Range of Recovery [%]Version 13.0-r

from86.3

90.4

96.4

to107.9

106.8

103.7

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10. LIMITATION 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.
10.1 Interfering Substances
Haemoglobin (up to 4 mg/ml), bilirubin (up to 0.125 mg/ml) and triglyceride (up to 30 mg/ml) have noinfluence on the assay results.
10.2 Drug Interferences
Until today no substances (drugs) are known to us, which have an influence to the measurement of Estriol ina sample.
10.3 High-Dose-Hook EffectNo hook effect was observed in this test.
11. REFERENCES / LITERATURE
Bashore, R.A., Westlake, J.R. Plasma unconjugated estriol values in high risk pregnancy.
Am. J. Obstet. Gynecol., June 15, 1977, p371-380

Symbols:

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Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContentCaution

Catalogue
numberEffective: 2020-01-17

Contains sufficient for<n> tests

For research use
only!
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