Instructions for use

Estradiol ELISAFR E-2000R

1.INTRODUCTION

1.1 Intended Use
The Estradiol ELISA is a competitive immunoassay for the quantitative measurement of estradiol in serumand plasma (EDTA).
2.PRINCIPLE
The Estradiol ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA), based on the principle ofcompetitive binding.
The microtiter wells are coated with an anti-Estradiol antibody. An unknown amount of estradiol present in thesample competes with an Estradiol-horseradish peroxidase conjugate for binding to the coated antibody. Afterincubation the unbound conjugate is washed off. The amount of bound peroxidase conjugate is inversely
proportional to the concentration of Estradiol in the sample. After addition of the substrate solution, theintensity of color developed is inversely proportional to the concentration of Estradiol in the sample.
3.WARNINGS AND PRECAUTIONS
1.

This kit is for research use only.

2.

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.

3.

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.

4.

Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for eachstep.

5.

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 colored. Do not pour reagents back into vials as reagent contamination may occur.

6.

Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells.

7.

Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.

8.

Allow the reagents to reach room temperature (21-26°C) before starting the test. Temperature will affectthe absorbance readings of the assay. However, values for the samples will not be affected.

9.

Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.

10. Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
11. Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagentsor specimens may give false results.
12. Handling should be done in accordance with the procedures defined by an appropriate national biohazard
safety guideline or regulation.
13. Do not use reagents beyond expiry date as shown on the kit labels.
14. All indicated volumes have to be performed according to the protocol. Optimal test results are only obtained
when using calibrated pipettes and microtiterplate readers.
15. 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.
16. Avoid contact with Stop Solution. It may cause skin irritation.
17. Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national
biohazard safety guideline or regulation.
18. For information please refer to Material Safety Data Sheets. Safety Data Sheets for this product areavailable upon request directly from the manufacturer.

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4.REAGENTS
4.1 Reagents provided
FR E-2031 Microtiterplate
12x8 (break apart) strips with 96 wells; Wells coated with anti-Estradiol antibody.
Standards
ready to use
Cat. no.

Standard

Concentration

Volume/Vial

FR E-2001

Standard A (0)0 pg/ml

0.3 mlFR E-2002

Standard B (1)25 pg/ml

0.3 mlFR E-2003

Standard C (2)75 pg/ml

0.3 mlFR E-2004

Standard D (3)225 pg/ml

0.3 mlFR E-2005

Standard E (4)675 pg/ml

0.3 mlFR E-2006

Standard F (5)2000 pg/ml

0.3 ml+FR E-2051 + FR E-2052 Control low / Control high2 vials, 0.3 ml each, ready to use; containing Estradiol in human serum-buffer matrix.

For control values and ranges please refer to QC-Datasheet
FR E-2040 Enzyme Conjugate
1 vial, 11 ml, ready to use; 17β-Estradiol labeled horseradish peroxidase in buffered matrix.
AR E-0055 Substrate Solution
1 vial, 22 ml each, ready to use; contains tetramethylbenzidine (TMB)
Hazards
identification:
H360D May damage the unborn child.
AR E-0080 Stop Solution
1 vial, 7 ml, ready to use; contains 2 N hydrochloric acid solutionHazards
identification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.
H335 May cause respiratory irritation.
AR E-0030 Wash Solution
1 vial, 50 ml (10X concentrated);see “Reagent Preparation”.
Note: Additional Standard A for sample dilution is available upon request.
4.2 Materials required but not provided

A microtiter plate reader capable for endpoint measurement at 450 nmCalibrated variable precision micropipettes (25 µl, 100 µl, 200 µl, 300 µl).
Microplate mixer operating more than 600 rpm
Absorbent paper
Distilled or deionized 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 be stable until expiration date. Do not use reagents beyond
this date. Opened reagents must be stored at 2°C -8°C. After first opening the reagents are stable for 30 days
if used and stored properly.
Microtiter wells must be stored at 2°C to 8°C. Take care that the foil bag is sealed tightly.

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4.4 Reagent preparation
Allow the reagents and the required number of wells to reach room temperature (21-26°C) before starting thetest.
Wash Solution:
Dilute 50 ml of 10X concentrated Wash Solution with 450 ml deionized water to a final volume of 500 ml.
The diluted Wash Solution is stable for at least 12 weeks at room temperature (21-26°C).
4.5 Disposal of the kits
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.
4.6 Damaged test kits
In case of any severe damage of the test kit or components, the manufacturer have to be informed in writing
within one week after receiving the kit. Severely damaged single components should not be used for a test run.
They have to be stored until a final solution has been found. After this, they should be disposed according tothe official regulations.
5. SPECIMEN
For determination of estradiol serum or plasma (EDTA) can be used. The procedure calls for25 µl sample per well. The samples should be assayed immediately or aliquoted and stored at ≤ -20°C. Avoid
repeated freeze-thaw cycles. Samples expected to contain estradiol concentrations higher than the highest
standard (2000 pg/ml) should be diluted with the Standard A before assay. The additional dilution step has tobe taken into account for the calculation of the results. Do not use grossly haemolytic, icteric or grossly
lipaemic specimens.
6. ASSAY PROCEDURE
6.1 General Remarks
All reagents and specimens must be allowed to come to room temperature before use. All reagents must be
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.
Respect the incubation times as stated in this instructions for use.






6.2 Assay procedure
Each run must include a standard curve.
1.

Prepare a sufficient number of microplate wells to accommodate standards and samples in duplicates.

2.

Dispense 25 µl of each Standard, Sample and Control with new disposable tips into appropriate
wells.

3.

Incubate for 60 minutes at room temperature on a plate shaker (> 600 rpm).

4.

Add 100 µl of Enzyme Conjugate into each well.

5.

Incubate for 60 minutes at room temperature on a plate shaker (> 600 rpm)
Important Note:
Optimal reaction in this assay is markedly dependent on shaking of the microplate!

6.

Discard the content of the wells and rinse the wells 4 times with diluted Wash Solution (300 µl perwell). Remove as much Wash Solution as possible by beating the microplate on absorbent paper.

7.

Add 200 µl of Substrate Solution to each well.

8.

Incubate without shaking for 30 minutes in the dark.

9.

Stop the reaction by adding 50 µl of Stop Solution to each well.

10.

Determine the absorbance of each well at 450 nm. It is recommended to read the wells within
15 minutes.

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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 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 and control, determine the corresponding concentrationfrom the standard curve.
4. Automated method: The results in the package insert have been calculated automatically using a 4 PL
(4 Parameter Logistics) curve fit. 4 Parameter Logistics is the preferred calculation method. Other data
reduction functions may give slightly different results.
5. The concentration of the samples can be determined directly from this standard curve. Samples withconcentrations higher than that of the highest standard have to be further diluted. For the calculation of theconcentrations, this dilution factor has to be taken into account.
Example of typical standard curve
Following data are intended for illustration only and should not be used to calculate results from another run.
Standard
Standard A
Standard B
Standard C
Standard D
Standard E
Standard F(0 pg/ml)(25 pg/ml)(75 pg/ml)(225 pg/ml)(675 pg/ml)(2000 pg/ml)

Optical Units(450nm)
2.738
2.439
1.999
1.320
0.711
0.346

7. Expected values
It is strongly recommended that each laboratory should establish its own normal ranges.
In a study conducted with apparently normal healthy adults, using the Estradiol ELISA the following values areobserved:Population
MalesFemales

Age

5%-95% Percentile< 50 years

n.d. – 36.2 pg/ml> 50 years

n.d. – 51.1 pg/ml< 50 years

n.d. – 111.4 pg/ml> 50 years

n.d. – 37.9 pg/ml

n.d.= non detectable
The values provided here were determined without consideration of the phase of menstrual cycle.
8. PERFORMANCE CHARACTERISTICS
8.1 Analytical Sensitivity
The lowest analytical detectable level of Estradiol that can be distinguished from Standard A is 6.2 pg/ml at the2SD confidence limit.

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8.2 Specificity (Cross Reactivity)
The following materials have been evaluated for cross reactivity. The percentage indicates cross reactivity at
50% displacement compared to Estradiol.
Steroid% Cross reaction

Androstenedione< 0.1

17-Hydroxyprogesterone< 0.1

Corticosterone< 0.1

Estriol

0.4

Estrone

4.2

Pregnenolone< 0.1

E2-3-Glucuronide

3.8

E2-3-Sulphate

3.6

E2-17-Glucuronide< 0.1

Progesterone< 0.1

Testosterone< 0.1

Fulvestrant

9.58.3 Assay dynamic range
The range of the assay is between 25 – 2000 pg/ml.
8.4 Reproducibility
8.4.1 Intra-Assay
The intra-assay variation was determined by 20 replicate measurements of three serum samples within one
run. The within-assay variability is shown below:
Serum 1

Serum 2

Serum 3

Mean (pg/ml)

83.8

269.9

794.8

SD

5.3

8.5

29.5

CV (%)

6.4

3.1

3.7

n=

20

20

20

8.4.2 Inter-Assay
The inter-assay (between-run) variation was determined by duplicate measurements of three serum samples in10 different tests.

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Serum 1

Serum 2

Serum 3

Mean (pg/ml)

85.2

251.3

727.8

SD

4.7

11.7

32.6

CV (%)

5.5

4.7

4.5

n=

1010

10

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8.5 Recovery
Using the standard matrix two spiking solutions of 100 ng/ml and 10 ng/ml were prepared. Three sera (1-3)
were spiked with 10 and 25 µl of the 10 ng/ml solution and with 6 µl of the 100 ng/ml solution leaving theserum matrices relatively intact. All samples were measured by the Estradiol ELISA procedure.
Sample1Spiking(pg/ml)-Measured(pg/ml)
27.8Expected(pg/ml)-Recovery
(%)
-

100

141.3

127.8

111%

250

315.0

277.8

113%

600

685.0

627.8

109%

-

10.3

-

-

100

119.3

110.3

108%

250

267.4

260.3

103%

600

710.3

610.3

116%

2

-

4.2

-

-

100

98.7

104.2

95%

250

267.3

254.2

105%

600

668.1

604.2

111%

3

8.6 Linearity
Three serum samples were assayed undiluted and diluted with Standard A.
Serum
12

3

Measured

Expected(pg/ml)(pg/ml)1 in 2
1 in 41 in 8

715.9
321.3
164.4
88.9./.
358.0
179.0
89.5./.
90%
92%
99%

1 in 2
1 in 41 in 81012.7553.3
306.0
146.8./.
506.4
253.2
126.6./.
109%
121%
116%

1 in 2
1 in 4

1099.2583.4
316.1./.
549.6
274.8./.
106%
115%

1 in 8

165.0

137.4

120%

Dilution

Linearity (%)

9. LIMITATIONS OF PROCEDURE
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
Any medication (cream, oil, pill etc.) containing Estradiol will significantly influence the measurement of thisanalyte.
The Estradiol ELISA should not be used for patients being treated with the drug fulvestrant (Faslodex®) whichcross reacts in the Estradiol ELISA and could lead to falsely elevated test results.
10. REFERENCES
1.
2.
3.
4.

Tsang, B.K., Armstrong, D.T. and Whitfield, J.F., Steroid biosyntheses by isolated human ovarianfollicular cells in vitro, J. Clin. Endocrinol. Metab. 51:1407 - 11 (1980).
Gore-Langton, R.E. and Armstrong, D.T., Follicular steroidogenesis and its control. In: The physiology ofReproduction, Ed.: Knobil, E., and Neill, J. et al., pp. 331-85. Raven Press, New York (1988).
Hall, P.F., Testicular Steroid Synthesis: Organization and Regulation. In: The Physiology of Reproduction,
Ed.: Knobil, E., and Neill, J. et al., pp 975-98. Raven Press, New York (1988).
Siiteri, P.K. Murai, J.T., Hammond, G.L., Nisker, J.A., Raymoure, W.J. and Kuhn, R.W., The serumtransport of steroid hormones, Rec. Prog. Horm. Res. 38:457 - 510 (1982).

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5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.

19.
20.
21.

Martin, B., Rotten, D., Jolivet, A. and Gautray, J-P-., Binding of steroids by proteins in follicular fluid of
the human ovary, J.Clin. Endicrinol. Metab. 35: 443-47 (1981).
Baird, D.T., Ovarian steroid secretion and metabolism in women. In: The Endocrine Function of theHuman Ovary. Eds.: James, V.H:T., Serio, M. and Giusti, G. pp. 125-33,
Academic Press, New York (1976).
McNastty, K.P., Baird, D.T., Bolton, A., Chambers, P., Corker, C.S. and McLean, H., Concentration ofoestrogens and androgens in human ovarian venous plasma and follicular fluid throughout the menstrual
cycle, J. Endocrinol. 71:77-85 (1976).
Abraham, G.E., Odell, W.D., Swerdloff, R.S. and Hopper, K., Simultaneous radioimmunoassay of plasma
FSH, LH, progesterone, 17-hydroxyprogesterone and estradiol-17ß during the menstrual cycle, J.Clin.
Endocrinol. Metab. 34:312-18 (1972).
March, C.M., Goebelsmann, U., Nakumara, R.M., and Mishell, D.R., Roles of oestradiol and progesterone
in eliciting midcycle luteinising hormone and follicle stimulating hormone surges.
J. Clin. Endicrinol. Metab. 49:507-12 (1979).
Simpson, E.R. and McDonald, P.C., Endocrinology of Pregnancy. In: Textbook of Endocrinology,
Ed.: Williams, R.H. pp412-22, Saunders Company, Philadelphia (1981).
Jenner, M.R., Kelch, R.P., et al., Hormonal Changes in prepubertal children, pubertal females and inprecocious puberty, premature thelarche, hypogonadism and in a child with feminising tumour,
J. Clin. Endocrinol. 34: 521 (1982).
Goldstein, D. et al., Correlation between oestradiol and progesterone in cycles with luteal phase
deficiency, Fertil. Steril. 37: 348-54 (1982).
Kirschner, M.A., The role of hormones in the etiology of human breast cancer, Cancer 39:2716 26(1977).
Odell, W.D. and Swerdloff, R.D., Abnormalities of gonadal function in men, Clin. Endocr. 8:149-80(1978).
McDonald, P.C., Madden, J.C., Brenner, P.F., Wilson, J.D. and Siiteri, P.K. Origin of oestrogen in normal
men and women with testicular feminisation, J.Clin. Endcrinol. Metabol. 49:905 (1979).
Peckham, M.J: and McElwain, T.J:, Testicular tumours, J.Clin. Endocrinol. Metab. 4:665-692 (1975).
Taubert, H.D. and Dericks-Tan, J.S.E., Induction of ovulation by clomiphene citrate in combination withhigh doses of oestrogens or nasal application of LH-RH. In: Ovulation in the Human. Eds.: Crosignandi,P.G. and Mishell, D.R., pp.265-73, Academic Press, New York (1976).
Fishel, S.B., Edwards, R.G., Purdy, J.M., Steptoe, P.C., Webster, J., Walters, E., Cohen, J. Fehilly, C.
Hewitt, J. and Rowland, G., Implantation, abortion and birth after in-vitro fertilisation using the natural
menstrual cycle or follicular stimulation with clomiphene citrate and human menopausal gonadotropin,
J. In Vitro Fertil. Embryo Transfer, 1:24-28 (1985).
Wramsby, H., Sundstorm, P. and Leidholm, P., Pregnancy rate in relation to number of cleaved eggs
replaced after in vitro-fertilisation of stimulating cycles monitored by serum levels of oestradiol andprogesterone as sole index. Human Reproduction 2: 325-28 (1987).
Ratcliff, W.A., Carter, G.D. et al., Estradiol assays: applications and guidelines for the provision of clinicalbiochemistry service, Ann. Clin. Biochem. 25:466-483 (1988).
Tietz, N.W., Textbook of Clinical Chemistry. Saunders, 1986.

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