Instructions for use

Estrone ELISAFR E-2300R

Estrone ELISA
1. INTRODUCTION
1.1 Intended use
The Estrone ELISA is an enzyme immunoassay for the quantitative measurement of Estrone in serum andEDTA Plasma.
2. PRINCIPLE OF THE TESTThe Estrone ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA), based on the principleof competitive binding.
The microtiter wells are coated with a polyclonal rabbit antibody directed towards an antigenic site of theEstrone molecule. Endogenous Estrone of a sample competes with an estrone-horseradish peroxidaseconjugate 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 Estrone in thesample. After addition of the substrate solution, the intensity of colour developed is inversely proportional tothe concentration of Estrone in the sample.
3 WARNINGS AND PRECAUTIONS
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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.
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.
The microplate contains snap-off strips. Unused wells must be stored at 2 °C - 8 °C in the sealed foil
pouch and used in the frame provided.
Pipetting of samples and reagents must be done as quickly as possible and in the same sequence foreach step.
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.
Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reusemicrowells.
Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
Allow the reagents to reach room temperature (21 °C - 24 °C) before starting the test. Temperature
will affect the absorbance readings of the assay. However, values for the samples will not be affected.
Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
Wear disposable latex gloves when handling specimens and reagents. Microbial contamination ofreagents or specimens may give false results.
Handling should be done in accordance with the procedures defined by an appropriate national
biohazard safety guideline or regulation.
Do not use reagents beyond expiry date as shown on the kit labels.
All indicated volumes have to be performed according to the protocol. Optimal test results are only
obtained when using calibrated pipettes and microtiterplate readers.
Do not mix or use components from kits with different lot numbers. It is advised not to exchange wellsof different plates even of the same lot. The kits may have been shipped or stored under differentconditions and the binding characteristics of the plates may result slightly different.
Avoid contact with Stop Solution containing 0.5 M H2SO4. It may cause skin irritation and burns.
Some reagents contain Proclin 300, BND and/or MIT as preservatives. In case of contact with eyes orskin, flush immediately with water.
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.
Chemicals and prepared or used reagents have to be treated as hazardous waste according to thenational biohazard safety guideline or regulation.
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-2331

Microtiterwells

Contents:

12x8 (break apart) strips, 96 wells,
Wells coated with anti-Estrone antibody (polyclonal).

Standards and Controls - Ready to use
Concentration pg/ml

Volume / Vial

FR E-2301

Cat. no.

Component

01 ml

FR E-2302

151 mlFR E-2303

301 mlFR E-2304

90

1 ml

FR E-2305

270

1 ml

FR E-2306

810

1 ml

FR E-2307

2400

1 ml

FR E-2351

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

1 ml

FR E-2352
Conversion:

pg/ml x 3.69 = pmol/lContents:

contain non mercury preservative.

1 ml

The standards are calibrated against certified reference material code E-075 (Cerilliant).
FR E-2340
Contents:
Volume:Enzyme Conjugate - Ready to useEstrone conjugated to horseradish peroxidase
contains non-mercury preservative
1 x 14 ml/vial
Substrate Solution - Ready to use

FR E-0055
Contents:

Tetramethylbenzidine (TMB)

Volume:1 x 14 ml
Stop Solution - Ready to use

FR E-0080
Contents:

contains 0.5 M H2SO4

Volume:1 x 14 ml/vial

Hazards
identification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.
Wash Solution - Concentrated 40x

FR E-0030
Volume:1 x 30 ml/vial
See “Reagent Preparation”

Note: Additional Standard A for sample dilution is available upon request.
4.2 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

4.3 Storage Conditions
When stored at 2 °C - 8 °C unopened reagents will retain reactivity until expiration date. Do not use
reagents beyond this date.
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Opened reagents must be stored at 2 °C - 8 °C. Microtiter wells must be stored at 2 °C - 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.
4.4 Reagent Preparation
Bring all reagents and required number of strips to room temperature (21 °C - 24 °C) 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 Sheet.
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 or EDTA 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.
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.
Plasma:Whole blood should be collected into centrifuge tubes containing anti-coagulant (e.g. Sarstedt Monovettewith the appropriate plasma preparation) and centrifuged immediately after collection.
5.2 Specimen Storage and Preparation
Specimens should be capped and may be stored for up to 4 days at 2 °C - 8 °C prior to assaying.
Specimens held for a longer time (up to 18 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 re-assayed 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 sample + 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
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All reagents and specimens must be allowed to come to room temperature before use. All reagents must
be mixed without foaming.
The test should not be performed above a temperature of 24 °C.
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 25 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 5 times with diluted Wash Solution (400 µl per well). Strike the wells sharply onabsorbent paper to remove residual droplets.
Important note: The sensitivity and precision of this assay is markedly influenced by the correct
performance of the washing procedure!

6.

Add 100 µl of Substrate Solution to each well.

7.

Incubate for 15 minutes at room temperature.

8.

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

9.

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.

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> 2400 pg/ml. For the calculation of the concentrations this dilution factor has to be taken into account.
6.3.1 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
Standard A (0 pg/ml)
Standard B (15 pg/ml)
Standard C (30 pg/ml)
Standard D (90 pg/ml)
Standard E (270 pg/ml)
Standard F (810 pg/ml)
Standard G (2400 pg/ml)

Optical Units (450 nm)
2.24
2.04
1.88
1.48
1.05
0.68
0.43

7. EXPECTED NORMAL VALUES
It is strongly recommended that each laboratory should determine its own normal and abnormal values.
In a study conducted with apparently normal healthy adults, using the Estrone ELISA the following values
are observed:
PopulationnMalesFemalespremenopausal
postmenopausal

60110
91

range (min. – max.)Mean(pg/ml)(pg/ml)9.0 - 79.1
47.6
11.2 - 338.3
9.4 - 59.6

69.4
28.6

2.5th - 97.5th
Percentile (pg/ml)
15.6 - 77.0

Median(pg/ml)
47.9

15.5 - 220.2
11.0 - 54.5

52.8
27.4

These results correlate well to published data (8, 9).
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8. 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 properperformance.
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 Report added to thekit. The values and ranges stated on the QC Report always refer to the current kit lot and should be used for
direct comparison 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 8.1 - 2400 pg/ml.
9.2 Specificity of Antibodies (Cross Reactivity)
The following substances were tested for cross reactivity of the assay:
CompoundCross Reactivity (%)

17-OH-Progesterone
Androstenedione
Corticosterone
Cortisone
DHEA
Estradiol
Estriol
Estrone 3-β-D-glucoronide
Estrone 3-sulfate
Ethisterone
Hydrocortisone
Progesterone
Testosterone

0.01
0.04
0.01
0.39
0.01
1.19
0.07
0.35
0.44
0.39
0.30
< 0.01
0.03

9.3 Sensitivity
The analytical sensitivity of the ELISA was calculated by subtracting 2 standard deviations from the mean of20 replicate analyses of the Zero Standard (Standard A) and was found to be 8.1 pg/ml.
The limit of detection (LoD) of the assay is 14.6 pg/ml.
The limit of quantification (LoQ) of the assay is 15.8 pg/ml.
9.4 Reproducibility
9.4.1 Intra Assay
The within assay variability is shown below:
Sample12
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n10
10
10
10

Mean (pg/ml)
57.68
61.00
82.14
409.13

Effective: 2019-04-03

CV (%)
8.4
6.7
6.8
6.4

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9.4.2 Inter Assay
The between assay variability is shown below:
Sample1234n30303030

Mean (pg/ml)
32.98
61.91
81.96
451.6

CV (%)
11.9
9.7
7.4
8.7

9.4.3 Inter-Lot
The inter-assay (between-lots) variation was determined by repeated measurements of 3 samples in 3
different kit lots.
Sample1234

n18181818Mean (pg/ml)
2005.491122.06
66.28
53.46

CV (%)
4.8
2.3
14.0
8.4

9.5 Recovery
Samples have been spiked by adding Estrone solutions with known concentrations in a 1:1 ratio.
The % recovery has been calculated by multiplication of the ratio of the measurements and the expectedvalues with 100 (expected value = (endogenous Estrone + added Estrone) / 2; because of a 1:2 dilution ofserum with spike material).
SampleSample Type
Concentration [pg/ml]
Average Recovery [%]Range of Recovery [%]

from
to1
Serum
31.95
102.0
96.0
104.2

from
to1
EDTA
92.42
110.3
108.4
112.1

2
EDTA
32.06
103.4
96.1
109.4

3
EDTA
33.83
93.3
85.7
110.2

4
Serum
46.08.
108.0
101.7
114.3

5
Serum
464.78
105.6
95.9
114.2

6Serum639.70
92.4
89.2
95.4

9.6 Linearity
SampleSample TypeConcentration [pg/ml]
Average Recovery
Range of Recovery (%)

2EDTA
210.70
101.4
95.7
105.9

3
Serum
496.00
106.0
88.9
113.4

4
Serum
547.96
96.6
85.0
109.0

5
Serum
1649.26
101.6
95.7
105.2

9.7 Comparison Studies
For method comparison, 40 samples spanning the complete assay range were tested by the Estrone ELISA
and by the RIA.
The resulting regression equation was:ELISA = 0.743 RIA + 15.52; R2 = 0.990
10. 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.
10.1 Interfering Substances
Haemoglobin (up to 4 mg/ml), Bilirubin (up to 0.5 mg/ml) and Triglyceride (up to 7.5 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 estrone
in a sample.
10.3 High-Dose-Hook EffectNo hook effect was observed in this test.
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11. REFERENCES / LITERATUR
1.

Resnik R, Killam AP, Battaglia FC et al: The stimulation of uterine blood flow by various estrogens.
Endocrinology. 1974; 94:1192,
2. Fayman C, Winter JSD, Reyes FI. Patterns of gonadotropins and gonadal steroids throughout life. Clin.
Obstet. Gynecol. 1976; 3:467-483.
3. Baird DT. Fraser IS. Blood production and ovarian secretion rates of estradiol-17ß and estrone inwomen throughout the menstrual cycle. J Clin Endocrinol. Metab. 1974; 38:1009-1017.
4. Lindbert BS, Johansson EDB, Nilsson BA: Plasma levels of non-conjugated oestrone, oestradiol-17b andoestriol during uncomplicated pregnancy. Acta Obstet Gynecol Scand. 1974; 32:21.
5. Drafta D, Schindler AE, Stroe EW, Neacsu E. Age-related changes of plasma steroids in normal adultmales. J Steroid Biochem. 1982; 17:683-687.
6. DeVane GW, Czekala NM, Judd HL, Yen SSC. Circulating gonadotropins, estrogens, and androgens inpolycystic ovarian disease. Am J Obstet Gynecol. 1975; 121:496.
7. Kushnir MM et al. High-sensitivity tandem mass spectrometry assay for serum estrone and estradiol.
Am J Clin Pathol. 2008; 129:530-9.
8. Jasuja GK et al. Age trends in estradiol and estrone levels measured using liquid chromatography
tandem mass spectrometry in community-dwelling men of the Framingham Heart Study. J Gerontol ABiol Sci Med Sci. 2013; 68:733-40.
9. Kaaks R, Lukanova A, Kurzer MS. Obesity, endogenous hormones, and endometrial cancer risk: asynthetic review. Cancer Epidemiol Biomarkers Prev. 2002; 11(12):1531-43.
10. Kuiper GG, et al. Comparison of the ligand binding specificity and transcript tissue distribution ofestrogen receptors alpha and beta. Endocrinology. 1997; 138:863–870.

Symbols:

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Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContentCaution

Catalogue
numberEffective: 2019-04-03

Contains sufficient for<n> tests

For research use
only!
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