Instructions for use

Progesterone Saliva ELISASA E-6300R

Free

Progesterone Saliva ELISA
1. INTRODUCTION
1.1 Intended Use
Enzyme immunoassay for the quantitative measurement of active free progesterone (a female hormone) insaliva.
1.2 Summary and Explanation
Progesterone (4-pregnene-3, 20-dione) is a C21 steroid hormone containing a keto-group (at C-3) and a doublebond between C-4 and C-5. Like other steroids, it is synthesized from cholesterol via a series of enzymemediated steps (1)The steroid hormone Progesterone is a female sex hormone which, in conjunction with estrogens, regulates theaccessory organs during the menstrual cycle and it is particularly important in preparing the endometrium forthe implantation of the blastocyte and in maintaining pregnancy (2)In non-pregnant women progesterone is mainly secreted by the corpus luteum whereas in pregnancy theplacenta becomes the major source (3,4). Minor sources for progesterone are the adrenal cortex for both sexes
and the testes for males.
The Progesterone level in saliva represents the concentration of the active free Progesterone.
2. PRINCIPLE OF THE TESTThe Progesterone Saliva 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 of theProgesterone molecule. Endogenous Progesterone of a sample competes with a Progesterone-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 Progesterone in thesample. After addition of the substrate solution, the intensity of colour developed is inversely proportional tothe concentration of Progesterone in the sample.
3. WARNINGS AND PRECAUTIONS
1.
2.

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 the
package 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 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 to 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, BND and MIT as preservatives. In case of contact with eyes or skin, flush
immediately with water.
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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.
4. REAGENTS
4.1 Reagents provided
Microtiterwells

SA E-6331
Contents:

12 x 8 (break apart) strips, 96 wells;
Wells coated with anti-Progesterone antibody (polyclonal).

Standards and Controls - Ready to use
Cat. no.

ComponentStandard

SA E-6301

Standard A

Concentration
0 pg/ml

Volume/Vial

SA E-6302

Standard B

10 pg/ml

1 ml

SA E-6303

Standard C

50 pg/ml

1 ml
1 ml

1 ml

Standard D

150 pg/ml

SA E-6305

Standard E

600 pg/ml

1 ml

SA E-6306

Standard F

2400 pg/ml

1 ml

SA E-6351

Control low

1 ml

SA E-6352

Control highControl values and ranges
please refer to vial label or QCReport.

SA E-6304

Conversion:

pg/ml x 3.18 = pmol/lContents:

Contain non-mercury preservative.

1 ml

Enzyme Conjugate - ready to use

SA E-6340
Contents:

Progesterone conjugated to horseradish peroxidase; Contain non‐mercury preservative.

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

SA E-6355
Contents:

Tetramethylbenzidine (TMB).

Volume:1 x 25 ml

FR E-0080

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

Contents:
Volume:Hazards
identification:

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

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
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A microtiter plate calibrated reader (450 nm / 620 - 630 nm)
Calibrated variable precision micropipettes (100 µl and 200 µl)
Absorbent paper
Distilled water
Timer (60 min range)
Semi logarithmic graph paper or software for data reduction

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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 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 prior to use.
Wash Solution
Add distilled water to the 40X concentrated Wash Solution.
Dilute 30 ml of concentrated Wash Solution with 1170 ml distilled 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 this productis 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, atthe latest, 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
to the official regulations.
5. SPECIMEN COLLECTION AND PREPARATION
Saliva can be used in this assay.
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).
If there is visible blood contamination of the donor specimen, it should be discarded, rinse the sampling device
with water, 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
Saliva samples should be collected using SALI-TUBES 100.
Do not use Salivette® tubes for sampling; this in most cases will result in significant interferences.
Other saliva sampling devices have not been tested and should be validated under the responsibility of theuser.
At least during the luteal phase of females there is a significant episodic excretion pattern of Progesterone. Dueto this episodic variation of the steroid secretion we highly recommend the strategy of multiple sampling.
In order to avoid arbitrary results we recommend that always 5 samples be taken within a period of 2 - 3 hours(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
Fresh saliva samples
Immediately after arrival in the lab fresh saliva samples should be frozen at least overnight at -20 °C.
Each saliva sample has to be frozen, thawed, and centrifuged in order to separate the mucins by centrifugation.
Storage:
immediately at -20 °C.
Then samples must be thawed and centrifuged for 5 to 10 minutes 10 000 g.
Thereafter, the clear supernatant must be transferred into a fresh tube.
Only this clear supernatant can be used as sample for the ELISA.
If a set of multiple samples has to be tested, the lab has to mix aliquots of the supernatant of the 5 single
samples in a separate sampling device and perform the testing from this mixture.
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Supernatant
Storage:
5 days at 2 °C to 8 °C
at least 5 days at -20 °C, in aliquots
The supernatant should be frozen only once.
Thawed supernatant 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 solution 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 Standard A (mix thoroughly)
10 µl of dilution a) + 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 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.

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
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

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

Secure the desired number of coated strips in the frame holder.

2.

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

3.

Dispense 200 µl of Enzyme Conjugate into each sample and standard 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 inverted 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 200 µl of Substrate Solution to each well.

7.

Incubate for 15 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 the solution in 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 absorbance obtained
3.

from each standard against its concentration with absorbance value on the vertical (Y) axis andconcentration on the horizontal (X) axis.
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 Instructions for Use have been calculated automatically using a
5.

4 Parameter curve fit. (4 Parameter Rodbard or 4 Parameter Marquardt are the preferred methods.) Other
data reduction functions may give slightly different results.
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 generation at the time of
assay.
Standard

Optical Units (450 nm)

Standard A (0 pg/ml)

1.96

Standard B (10 pg/ml)

1.72

Standard C (50 pg/ml)

1.41

Standard D (150 pg/ml)

1.05Standard E (600 pg/ml)

0.58

Standard F (2400 pg/ml)

0.23

7. EXPECTED NORMAL VALUES
It is strongly recommended that each laboratory should determine its own normal and abnormal values.
In order to determine the normal range of Progesterone Saliva ELISA, saliva samples from 50 adult male and
120 female apparently healthy subjects, ages 21 to 75 years, were collected in the morning and analyzed using
the Progesterone Saliva ELISA kit.
The following ranges were calculated from this study.

Age group
Women

21 - 50 years
21 - 50 years
51 - 75 years

Men

Salivary progesterone pg/ml
Follicular phasen = 40
Luteal phasen = 40
Postmenopausal
n = 40n = 5019.6 - 86.5 pg/ml
99.1 - 332.6 pg/ml
6.0 - 56.4 pg/ml
1.1 - 44.4 pg/ml

The values differ between age, new born, children, adolescents and adults.
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 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 Report 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 directcomparison 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 the
manufacturer directly.
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9. PERFORMANCE CHARACTERISTICS
9.1 Assay Dynamic Range
The range of the assay is between 1.1 – 2400 pg/ml.
9.2 Specificity
The following materials have been evaluated for cross reactivity. The percentage indicates cross-reactivity at
50% displacement compared to Progesterone.
Steroid% Cross-reactivity

Progesterone
Desoxycorticosterone
Pregnenolone
17α-Hydroxyprogesterone
Corticosterone
11-Desoxycortisol
Estriol
Estradiol 17β
Testosterone
Cortisone
DHEA-S
Cortisol
Androstendione
DHEA
Estron

100.0
1.1
0.35
0.9
0.2
0.1
0.0
0.0
0.2
< 0.1
0.0
2.6
0.4
0.0
0.0

9.3 Sensitivity
The analytical sensitivity of the Progesterone Saliva ELISA was calculated by subtracting 2 standard deviations
from the mean of 20 replicate analyses of the Standard A and was found to be 1.1 pg/ml
9.4 Reproducibility
Intra-Assay
The within assay variability is shown below:
SamplenMean (pg/ml)
CV (%)
1
20
185.7
8.1
2
20
353.6
5.5
3
20
625.3
5.3
Inter-Assay
The between assay variability is shown below:
SamplenMean (pg/ml)
CV (%)
1
20
109.7
10.7
2
20
137.8
10.1
3
20
1945.8
6.7
Inter-Lot
The inter-assay (between-lots) variation was determined by repeated measurements of 3 samples in 3 different
kit lots.
The between lot variability is shown below:
Sample
1
2
3

n
18
18
18

Mean (pg/ml)
196.6
346.9
612.4

CV (%)
5.4
3.4
3.9

9.5 Recovery
Recovery of the Progesterone Saliva ELISA was determined by adding increasing amounts of the analyte tothree different saliva samples containing different amounts of endogenous analyte. Each sample (native andVersion 8.0-r

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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.
Concentration (pg/ml)
Average Recovery (%)
Range of Recovery (%)

from
to

Sample 1
28.6
102.8
89.1
113.1

Sample 2
261.0
98.6
93.8
107.5

Sample 3
378.0
99.7
93.6
105.1

9.6 Linearity
In total six saliva samples containing different amounts of analyte were serially diluted with Standard A andassayed with the Progesterone Saliva ELISA.
Three of these samples were serially diluted directly, and the other 3 samples at first were spiked withprogesterone and then serially diluted up to 1:128.
The percentage recovery was calculated by comparing the expected and measured values for progesterone.
Samples above this range must be diluted and re-run.
Concentration (pg/ml)
Average Recovery (%)
Range of Recovery
(%)

from
to

Sample 1
130.5
98.9
85.7
111.6

Sample 2
321.0
107.1
102.9
112.1

Sample 3
378.7
104.6
95.3
109.8

Sample 4
501.6
100.0
89.3
105.3

Sample 5
580.0
98.0
86.9
109.7

Sample 61000.0
90.6
86.0
97.3

9.7 Comparison Studies

Progesterone Saliva ELISA (pg/ml)

A study was performed that evaluated 24 saliva samples collected from adult men and women. The samples
were analyzed with the Progesterone Saliva ELISA and a LC-MS/MS mass spectrometer to determine theconcentration of free progesterone in the saliva samples. A correlation of 0.997 and regression formula ofy = 0.9612x - 11.071 were obtained.

LC-MS/MS (pg/ml)

10. 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.
10.1 Interfering Substances
Blood contamination ≥ 0.16% in saliva samples will affect results, and usually can be seen by eye.
Concentrations of Sodium Azide ≥ 0.02% interferes in this assay and may lead to false results.
10.2 High-Dose-Hook EffectNo hook effect was observed in this test.
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11. REFERENCES / LITERATURE
1.
2.

3.

4.

Miller W.L. (1988): Molecular biology of steroid hormone synthesis,
Endocrin. Rev. 9, 295-318
Filicori M., Butler J.P., Crowley W.F. Jr. (1984): Neuroendocrine regulation of the corpus luteum in thehuman,
J. Clin. Invest. 73, 1638
Csapol Al., Pulkkinen M. O., Wiest W.G. (1973): Effects of lutectomy and progesterone replacement
therapy in early pregnancy patients.
Am J. Obstet. Gynecol. 115, 759
Henson M.C. (1998): Pregnancy maintenance and the regulation of placental progesterone biosynthesis in
the baboon.
Human Reproduction Update, 4, 389-405

Symbols:

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Consult instructions
for useContent

CautionCatalogue
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Contains sufficient for<n> tests

For research use
only!
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