Instructions for use

Estradiol Saliva ELISASA E-6200R

Free

Estradiol Saliva ELISA
1. INTRODUCTION
1.1 Intended Use
Enzyme immunoassay for the quantitative measurement of active free Estradiol, an estrogenic steroid, insaliva. Results may be used in research of various hormonal sexual disorders and in assessing placental
function in complicated pregnancy. It is intended only for research use.
1.2 Summary and Explanation
Estradiol (1,3,5(10)-estratriene-3,17β-diol; 17β-estradiol; E21) is a C18 steroid hormone with a phenolic
A ring. This steroid hormone has a molecular weight of 272.4. It is the most potent natural Estrogen,produced mainly by the Graffian follicle of the female ovary and the placenta, and in smaller amounts by theadrenals, and the male testes (1-3).
Estradiol (E2) is secreted into the blood stream where 98% of it circulates bound to sex hormone binding
globulin (SHBG) and to a lesser extent to other serum proteins such as albumin. Only a small fraction
circulates as free hormone or in the conjugated form (4,5). Estrogenic activity is effected via estradiolreceptor complexes which trigger the appropriate response at the nuclear level in the target sites. Thesesites include the follicles, uterus, breast, vagine, urethra, hypothalamus, pituitary and to a lesser extent theliver and skin.
In non-pregnant women with normal menstrual cycles, estradiol secretion follows a cyclic, biphasic patternwith the highest concentration found immediately prior to ovulation (6, 7). The rising estradiol concentrationis understood to exert a positive feedback influence at the level of the pituitary where it influences thesecretion of the gonadotropins, follicle stimulating hormone (FSH), and luteinizing hormone (LH), which areessential for follicular maturation and ovulation, respectively (8). Following ovulation, estradiol levels fall
rapidly until the luteal cells become active resulting in a secondary gentle rise and plateau of estradiol in theluteal phase. During pregnancy, maternal serum Estradiol levels increase considerably, to well above thepre-ovulatory peak levels and high levels are sustained throughout pregnancy (9).
2. PRINCIPLE OF THE TESTThe Estradiol Saliva ELISA kit is based on the competition principle and the microplate separation.
An unknown amount of Estradiol present in the sample and a fixed amount of Estradiol conjugated withhorseradish peroxidase compete for the binding sites of a polyclonal Estradiol antiserum coated onto thewells.
After two hours incubation the microtiter plate is washed to stop the competition reaction. Having added thesubstrate solution the concentration of Estradiol is inversely proportional to the optical density measured.
3. WARNINGS AND PRECAUTIONS
1.
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14.

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 of the
package insert provided with the kit. Be sure that everything is understood.
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.
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 to 26 °C) before starting the test. Temperature
will affect the absorbance readings of the assay. However, values for the patient samples will not beaffected.
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.

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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 wellsof different 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, BND and 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 thenational 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.
4. REAGENTS
4.1 Reagents provided
Microtiterwells

SA E-6231
Contents:

12 x8 (break apart) strips, 96 well;
Wells coated with a anti-Estriol antibody (polyclonal).

Standards and Controls- ready to use

SA E-6201

0

Volume /Vial
1 ml

SA E-6202

1

1 ml

SA E-6203

5

1 ml

SA E-6204

10

1 ml

SA E-6205

50

1 ml

SA E-6206
SA E-6251

100
Control values and ranges
please refer to vial label
or QC-Datasheet

1 ml
1 ml

Cat. noComponent

SA E-6252
Contents:

1 ml

Contain non-mercury preservative.
Enzyme Conjugate - Ready to use

SA E-6240
Contents:

Concentration [pg/ml]

Estradiol conjugated to horseradish peroxidase
Contains non-mercury preservative.

Volume:1 x 26 ml
Substrate Solution - Ready to use

SA E-0055
Contents:

Tetramethylbenzidine (TMB ).

Volume:1 x 25 ml
Stop Solution - Ready to use

FR E-0080
Contents:

0.5 M H2SO4
Avoid contact with the stop solution. It may cause skin irritations and burns.

Volume:1 x 14 ml

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

FR E-0030
Volume:1 x 30 ml
Concentrate for 1200 ml.

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4.2 Material required but not provided
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Calibrated EIA reader adjusted to read at 450 nm
Calibrated variable precision micropipettes (100 µl and 200 µl)
Distilled or Deionized water
0.9 % NaCl solution
Timer (60 min range)
Reservoirs (disposable)
Test tube or microtube rack in a microplate configuration
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 two months if stored as described above.
4.4 Reagent Preparation
Bring all reagents to room temperature before 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 Material 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
Eating, drinking, chewing gums or brushing teeth should be avoided for 30 minutes before sampling.
Otherwise, it is recommended to rinse mouth thoroughly with cold water 5 minutes prior to sampling.
Do not collect samples when oral diseases, inflammation or lesions exist (blood contamination).
In case of visible blood contamination the patient should discard the sample, rinse the sampling device withwater, wait for 10 minutes and take a new sample.
Note: Samples containing sodium azide should not be used in the assay.
5.1 Specimen Collection
It is recommended to collect saliva samples with commercially available equipment (e.g. Sali Set, catalogue
no. SA D-6100, 100 pieces).
Do not use any cotton swab for sampling, such as Salivettes; this in most cases will result in significant
interferences.
Due to the episodic secretion pattern of steroid hormones it is important to care for a proper timing of thesampling.
In order to avoid arbitrary results we recommend that always 5 samples be taken within a period of 2 - 3hours (multiple sampling) preferably before a meal.
As food might contain significant amounts of steroid hormones samples preferably should be taken while
fasting. If fasting should be a problem the collection period should be timed just before lunch or before
dinner.
5.2 Specimen Storage and Preparation
Specimens should be capped and may be stored for up to one week at 4 °C prior to assaying.
Specimens held for a longer time should be frozen -20 °C prior to assay. Even repeated thawing andfreezing is no problem.
Each sample has to be frozen, thawed, and centrifuged at least once anyhow in order to separate the mucins
by centrifugation.
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Upon arrival of the samples in the lab the samples have to stay in the deep freeze at least overnight. Next
morning the frozen samples are warmed up to room temperature and mixed carefully.
Then the samples have to be centrifuged for 5 to 10 minutes (at 3000 - 2000 × g).
Now the clear colorless supernatant is easy to pipette.
If a set of multiple samples have to be tested, the lab (after at least one freezing, thawing, andcentrifugation cycle) has to mix the aliquots of the 5 single samples in a separate sampling device andperform the testing from this mixture.
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 0.9 % NaCl 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 saliva + 90 µl 0.9 % NaCl (mix thoroughly)
10 µl of dilution a + 90 µl 0.9 % NaCl (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.
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.

6.2 Test Procedure
Each run must include a standard curve.
All standards, samples, and controls should be run in duplicate. All standards, samples, and controls should
be run concurrently so that all conditions of testing are the same.
1.

Secure the desired number of Microtiter wells in the holder.

2.

Dispense 100 µl of each Standard, Control and samples with new disposable tips into appropriate
wells.

3.

Incubate for 30 minutes at room temperature4.

Dispense 200 µl of Enzyme Conjugate into each sample and standard well
Mix the plate thoroughly for 10 seconds. It is important to have a complete mixing in this step.

5.

Incubate for 120 minutes at room temperature.

6.

Briskly shake out the contents of the wells.
Rinse the wells 3 times with diluted Wash Solution (400 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 the washing procedure!

7.

Add 200 µl of Substrate Solution to each well.

8.

Incubate for 30 minutes at room temperature.

9.

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

10.

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 patient 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.
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4. Automated method: The results in the IFU have been calculated automatically using a 4 PL (4 Parameter
Logistics) curve fit. 4 Parameter Logistics is the preferred method. 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> 100 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 of
assay.
Standard
Standard A
Standard B
Standard C
Standard D
Standard E
Standard F

Absorbance Units(0 pg/ml)(1 pg/ml)(5 pg/ml)(10 pg/ml)(50 pg/ml)(100 pg/ml)

1.89
1.71
1.48
1.20
0.46
0.32

7. EXPECTED NORMAL VALUES
In order to determine the normal range of Salivary Estradiol, 18 saliva samples from adult male and 54
female apparently healthy subjects, ages 19 to 75 years, were collected in the morning and analyzed using
the Estradiol Saliva ELISA kit.
The following ranges were calculated from this study.
Age group
Women
Men

19 – 50 yrs.
51 - 75 yrs.
18 - 75 yrs.

Estradiol Saliva [pg/ml]
n = 41
Postmenopausal: n = 13n = 18

0.6 – 6.3
0.6 – 3.1
0.6 – 3.1

Salivary Estradiol values show a clear circadian rhythm. We therefore recommend the saliva samples be
obtained the same hour each day.
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 to
the 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 patient 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 the
manufacturer directly.
9. PERFORMANCE CHARACTERISTICS
9.1 Assay Dynamic Range
The range of the assay is between 0.6 – 100 pg/ml.

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9.2 Specificity
The following materials have been evaluated for cross reactivity. The percentage indicates cross reactivity at
50% displacement compared to Estradiol.
Compound% Cross reactivity

Compound% Cross reactivity

Estradiol-17ß
Androstenedione
Androsterone
Corticsterone
Cortisone
Epiandrosterone
16-Epiestriol
Estradiol-3-sulfate
Estradiol-3-glucoronide
Estradiol-17α
Estriol
Estriol-16-glucoronide
EstroneEstrone-3-sulfate
Dehydroepiandrosterone

100
0
0
0
0
0
0
0
0
0
2.27
0
6.86
0
0

11-Deoxycortisol
21-Deoxycortisol
Dihydrotestosterone
Dihydroepiandrosterone
20-Dihydroprogesterone
11-Hydroxyprogesterone
17α-Hydroxyprogesterone
17α-Pregnenolone
17α-Progesterone
Pregnanediol
Pregnanetriol
Pregnenolone
Progesterone
Testosterone
Fulvestrant

0
0
0
0
0
0
0.003
0
0
0
0
0
0
0.033
0.9

9.3 Sensitivity
The lowest detectable level of Estradiol that can be distinguished from the Zero Standard is 0.6 pg/ml at the
95 % confidence limit.
9.4 Reproducibility
9.4.1 Intra Assay
The intra-assay variation was determined by 20 replicate measurements of three saliva samples Estradiol
Saliva ELISA kit.
The within assay variability is shown below:

Mean
SD (pg/ml)
CV (%)
n=

Sample 1

Sample 2

Sample 3

8.3
0.7
8.3
20

30.5
0.7
2.4
20

21.4
0.7
3.2
20

9.4.2 Inter Assay
The inter-assay (between-run) variation was determined by duplicate measurements of four saliva samples
over 10 different days runs.
The between assay variability is shown below:
Mean
SD (pg/ml)
CV (%)
n=

Sample 1

Sample 2

Sample 3

6.6
0.8
12.0
40

29.9
1.5
4.9
40

65.0
1.8
2.8
40

9.5 Recovery
Recovery of the Estradiol Saliva ELISA was determined by adding increasing amounts of the analyte to threedifferent saliva samples containing different amounts of endogenous analyte. Each sample (non-spiked andspiked) 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.
Concentration (pg/ml)
Average % recovery
Range offrom
Recovery%toVersion: 13.0-r

Sample 1
0.6
98.9
86.6
112.0

Sample 2
19.8
90.1
85.4
96.1

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Sample 3
20.8
106.3
102.4
111.0

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9.6 Linearity
Three samples (saliva) containing different amounts of analyte were serially diluted up to 1:16 with
0.9% NaCl and assayed with the Estradiol Saliva ELISA. The percentage recovery was calculated bycomparing the expected and measured values for Saliva estradiol.
Concentration (pg/ml)
Average% recovery
Range offrom
Recovery%toSample 1
75.6
104.8
96.0
112.8

Sample 2
62.9
95.7
88.5
106.7

Sample 3
31.7
91.6
90
93.3

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.
The patient should not eat, drink, chew gum or brush teeth for 30 minutes before sampling. Otherwise rinse
mouth thoroughly with cold water 5 min prior to sample collection. Do not collect samples when oraldiseases, inflammation or lesions exist (blood contamination).
10.1 Interfering Substances
Blood contamination of more than 0.16 % in saliva samples will affect results, and usually can be seen byeye. Therefore, samples containing any visible blood should not be used.
Concentrations of Sodium Azide ≥ 0.02 % interferes in this assay and may lead to false results.
10.2 Drug Interferences
The Estradiol Saliva ELISA should not be used for patients being treated with the drug fulvestrant(Faslodex®) which cross reacts in the Estradiol Saliva ELISA and could lead to falsely elevated test results.
10.3 High-Dose-Hook EffectNo hook effect was observed in this test.
11. REFERENCES / LITERATURE
1. Tsang B.K., et al. (1980) Steroid biosyntheses by isolated human ovarian follicular cells in vitro,
J. Clin. Endocrinol. Metab. 51, 1407 – 11
2. Gore-Langton R. E. et al. (1988): Follicular steroidogenesis and its control. In: The physiology of
Reproduction.
ED.: Knobil et al. pp.331-385, Raven press, New York3. Hall, P.F. (1988): Steroid synthesis: Organization and Regulation. In: The Physiology of Reproduction.
Ed.: Knobil et al., pp 975 – 988, Raven press, New York
4. Siiteri
P.K.
et
al.
(1982):
The
serum
transportofsteroidhormones.
Rec. Progr. Horm. Res. 38, 457 – 510
5. Martin B. et al. (1981): Binding of steroids by proteins in follicular fluid of the human ovary.
J. Clin. Endocrinol. Metab. 35, 443 – 447
6. Baird D.T. (1976): Ovarian steroid secretion and metabolism in women. In: The Endocrine function of
the human ovary. Eds.: James V.H.T., Serio M. and Guisti G., pp. 125 – 33, Academic press, New York7. McNtty K.P. et al. (1976): Concentration of estrogens and androgens in human ovarian venous plasma
and follicular fluid throughout the menstrual cycle. J. Endocrinol. 71, 77 –85
8. March C.M. et al. (1979): Roles of estradiol and progesterone in eliciting mid-cycle luteinizing hormone
and follicle stimulating hormone surges. J. Clin. Endocrinol. Metab. 49, 507 – 12
9. Simpson E.R. and McDonald P.C. (1981): Endocrinology of pregnancy. In: Textbook of Endocrinology,
Ed.: Williams R.H. pp. 412 – 22, Saunders Company, Philadelphia
Symbols:

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Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContent

CautionCatalogue
numberEffective 2018-05-30

Contains sufficient for<n> tests

For research use
only!
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