Instructions for use

17-OH-Progesterone Saliva ELISASA E-6400R

Free

17-OH-Progesterone Saliva ELISAINTRODUCTION
Intended Use
An enzyme immunoassay for the quantitative measurement of active free 17-hydroxyprogesterone in saliva.
PRINCIPLE OF THE TESTThe Salivary 17α-OHP 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 antibody (rabbit) directed towards an antigenic site on the
17α-OHP molecule. Endogenous 17α-OHP of a sample competes with a 17α-OHP-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 17α-OHP in thesample. After addition of the substrate solution, the intensity of colour developed is inversely proportional tothe concentration of 17α-OHP in the sample.
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 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 (21-26°C) before starting the test. Temperature will affectthe 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.
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 Material Safety Data Sheets.
Material Safety Data Sheets for this product are available upon request directly from the manufacturer.

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REAGENTS
Reagents provided
SA E-6431 Microtiterwells
12x8 (break apart) strips, 96 wells; Wells coated with a anti-cortisol antibody (polyclonal).
Standards
Cat. no.

Standard

SA E-6401

Standard A (0)SA E-6402
SA E-6403

Concentration

Volume/Vial

0 pg/ml

1 ml

Standard B (1)10 pg/ml

1 ml

Standard C (2)50 pg/ml

1 ml

SA E-6404

Standard D (3)250 pg/ml

1 ml

SA E-6405

Standard E (4)500 pg/ml

1 ml

SA E-6406

Standard F (5)1000 pg/ml

1 ml

SA E-6451

Control low

1 ml

SA E-6452

Control highRefer to vial labels
or QC-Datasheet!

1 ml

Conversion: pg/mL x 3.03 = pmol/L* contain 0.03% Proclin 300, 0.015% BND and 0.010% MIT as preservative.
SA E-6440 Enzyme Conjugate
1 vial, 26 mL, ready to use; 17α-OHP conjugated to horseradish Peroxidase;* contain 0.03% Proclin 300, 0.015% BND and 0.010% MIT as preservative.
SA E-6455 Substrate Solution
1 vial, 25 mL, ready to use; Tetramethylbenzidine (TMB)
Hazards
identification:
H360D May damage the unborn child.
FR E-0080 Stop Solution
1 vial, 14 mL, ready to use; contains 0.5M H2SO4.
Avoid contact with the stop solution. It may cause skin irritations and burns.
MöglicheGefahren:
H290 Kann gegenüber Metallen korrosiv sein.
H314 Verursacht schwere Verätzungen der Haut und schwere Augenschäden.
FR E-0030 Wash Solution
1 vial, 30 mL (40X concentrated); see „Preparation of Reagents“.
* BND = 5-bromo-5-nitro-1,3-dioxane
MIT = 2-methyl-2H-isothiazol-3-one
Note: Additional Standard A for sample dilution is available upon request.
Material required but not provided
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A microtiter plate calibrated reader (450 ± 10 nm).
Calibrated variable precision micropipettes (25 µL, 100 µL, 200 µL, 250 µL).
Absorbent paper.
Distilled or deionized water
Timer (60 min. range).
Semi-logarithmic graph paper or software for data reduction

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.

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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 six weeks if
stored as described above.
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.
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 Material Safety Data Sheet.
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.
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 discard the sample, 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.
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 – 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.
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 and freezing is
no problem.
Each sample has to be frozen, thawed, and centrifuged at least once anyhow in order to separate the mucins by
centrifugation.
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 x 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, and centrifugation
cycle) has to mix the aliquots of the 5 single samples in a separate sampling device and perform the testingfrom this mixture.

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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:
10 µl saliva + 90 µl Standard A (mix thoroughly)b) Dilution 1:100:
10 µl of dilution a + 90 µl Standard A (mix thoroughly).
ASSAY PROCEDURE
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.

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 25 µL of each 17α-OHP Standard, Control and samples with new disposable tips into appropriate
wells.
3. Dispense 250 µL Enzyme Conjugate into each well.
Mix the plate thoroughly 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 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!
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 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.

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 determine the corresponding concentration from thestandard curve.
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 giveslightly different results.
5. The concentration of the samples can be read directly from this standard curve. Samples with concentrations
higher than that of the highest standard have to be further diluted or reported as > 1000 pg/mL. For the
calculation of the concentrations this dilution factor has to be taken into account.

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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

Optical Units (450 nm)

Standard A (0 pg/mL)Standard B (10 pg/mL)Standard C (50 pg/mL)Standard D (250 pg/mL)Standard E (500 pg/mL)Standard F (1000 pg/mL)

1.92
1.72
1.47
0.91
0.64
0.41

EXPECTED NORMAL VALUES
In order to determine the normal range of SLV 17α -OHP, two studies were performed using saliva samples
from 129 apparently healthy children ages 6 – 12 years, 132 males ages 21 to 70 years, and 252 females of
non-pregnant women with regular menstrual cycles, ages 21 to 50 years. Saliva samples were collected in themorning, frozen at -20C, and analyzed using the SLV 17α-OHP ELISA kit.
The following ranges were calculated from this study.
Summary Normal ranges for Salivary 17α-OHP ELISAAge group
MeanChildren
WomenMen

6 – 12 yrs

N = 129

21 – 50 yrs

Follicular phase:
N = 124
Luteal phase:
N = 128

21 – 70 yrs

N = 152

S.D.

Range 5 - 95%

16.9 pg/mL

9.5 pg/mL

3.0 – 32.9 pg/mL

22.0 pg/mL

11.1 pg/mL

8.2 – 41.1 pg/mL

51.2 pg/mL

17.3 pg/mL

28.1 – 84.8 pg/mL

24.9 pg/mL

12.6 pg/mL

10.6 – 54.8 pg/mL

We recommend that each laboratory determine its own range for the population tested.
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 certificate added to thekit. 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 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.
PERFORMANCE CHARACTERISTICS
Assay Dynamic Range
The range of the assay is between 3.6 – 1000 pg/mL.

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Specificity of Antibodies (Cross Reactivity)
The following materials have been evaluated for cross reactivity. The percentage indicates cross reactivity at
50% displacement compared to 17α-OHP.
Steroid% Cross reaction

17- α -OH Progesterone

100%

Estriol< 0.01

Estradiol 17β< 0.01

Testosterone< 0.01

Dihydrotestosterone< 0.01DOC

0.05

11-Desoxycortisol

1.40

Progesterone

1.20

DHEA

< 0.01

DHEA-S< 0.001

Cortisol< 0.01

Corticosterone< 0.05

Aldosterone< 0.01Androstenedione< 0.01Dehydroepiandrosten sulfate< 0.01

Prednisone< 0.01

Sensitivity
The analytical sensitivity of this ELISA was calculated by subtracting 2 standard deviations from the mean of 20
replicate analyses of the Zero Standard (Standard A).
Analytical sensitivity is: 2.5 pg/mL
Functional sensitivity of the assay is : 3.6 pg/mL
Reproducibility
Intra-Assay
The intra-assay reproducibility was determined by replicate measurements of 6 saliva samples using the ELISA
kit. The within assay precision is shown below:
sample 4

sample 3

sample 1

sample 2

Sample 6

sample 5

Mean (pg/mL)

24.2

37.5

63.3

90.3

447.4

820.4

S.D.

1.9

1.4

4.6

4.5

17.8

29.0

CV (%)

8.0

3.6

7.3

5.0

4.0

3.5

n=

20

20

20

20

20

20

Inter-Assay
The inter-assay variation was determined by duplicate measurements of six saliva samples on 10 different days
using SLV 17-OHP ELISA kits. Mean of the duplicate measurements is given below.

Mean (pg/mL)SD
CV (%)
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sample 4

sample 1

sample 5

sample 2

sample 3

Sample 6

9.4
1.0
10.4
20

50.7
1.7
3.4
20

101.1
7.6
7.5
20

211.8
8.3
3.9
20

512.5
10.3
2.0
20

800.2
38.2
4.8
20

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Inter-Lot
The inter-assay (between-lots) variation was determined by repeated measurements of six saliva samples inthree different kit lots.
sample 4

sample 1

sample 5

sample 2

sample 3

sample 6

10.2
1.3
12.4
9

44.3
1.5
3.4
9

100.4
6.1
6.1
9

205.3
3.6
1.8
9

515.7
19.5
3.8
9

798.1
46.8
5.9
9

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

RecoveryRecovery of this ELISA was determined by adding increasing amounts of the analyte up to 500 pg/mL 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
tosaliva 2

saliva 3

saliva 1

saliva 4

saliva 5

saliva 6

3.7
104.1
97.7
103.9

17.9
102.1
96.6
107.5

282.8
101.7
98.2
109.5

824.1
98.3
93.1
103.8

1200.0
100.4
94.5
105.2

1050.0
99.3
93.7
103.5

Linearity
Six saliva samples containing different amounts of analyte (spiked and unspiked) were serially diluted with zerostandard and assayed with this ELISA. Three native samples were serially diluted up to 1:128, and 3 samples
were spiked with 17α-OHP and then serially diluted up to 1:10. The percentage recovery was calculated bycomparing the expected and measured values for 17α-OHP.
An assay linearity of 3.6 – 1000 pg/mL has been identified as the usable range. Samples above this range
must be diluted and re-run.
sample 2

sample 1

sample 3

sample 6

sample 5

sample 4

pg/mL

39.3

96.6

194.9

816.6

1050.01200.0

Average % Recovery
Range of
from

99.3
87.2

95.0
86.1

100.5
91.5

97.6
93.8

99.3
93.7

100.4
80.0

% Recovery

107.4

99.0

113.2

103.5

103.5

105.2

Concentration

toComparison Studies

E L I S A (pg/ml)

A study was performed using non spiked Saliva from adult normal and CAH-persons were assayed in parallelwith this ELISA and a reference LC-MS. Seventy-six saliva samples were tested on both the LC-MS and the SLVELISA test. The following results were obtained.

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ELISA

Another study was performed using 60 serum and saliva samples were taken in parallel at 8:00 am andassayed using the Salivary 17-OHP ELISA for the saliva samples and a Coated Tube RIA for 17-OHP for the sera
samples. The persons were a collective of 22 children and young adults suffering from congenital adrenal
hyperplasia (CAH). The results cover several years.

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 person 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 oral diseases,
inflammation or lesions exist (blood contamination).
Interfering Substances
Blood contamination of more than 0.08% in saliva samples will affect results, and usually can be seen by eye.
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.
High-Dose-Hook EffectNo hook effect was observed in this test.
REFERENCES / LITERATURE
1.

2.
3.

4.
5.
6.
7.

Chrousos G. P., Loriaux D. L., Mann D. L. et al. : Late onset 21-hydroxylase deficiency mimicking
idiopathic hirsutism or polycystic ovarian disease.
Annals Intern. Med. 96: 143, 1982.
Meikle A. W.,Worley R. J., West C. D.: Adrenal corticoid hyper-response in hirsute women.
Fertil. Steril. 41: 575, 1984
Bustler J. E., Chang R. J., Preston D. L., et al. : Interrelationships of circulating maternal steroids ;progesterone, 16-α hydroxyprogesterone, 17-α hydroxyprogesterone, 20-α hydroxyprogesterone, gamma5-pregnenolone.
J. Clin. Endocrinol. Metab. 48: 133, 1979
Urban M. D., Lee P. A., Mignon C. J.: Adult high infertilità in men with congenital adrenalized hyperplasia.
N. Engl. J. Med. 299: 1392, 1978
Zerah, M., Pang, S., and New, M Morning Salivary 17-Hydroxyprogesterone Is a Useful Screening Test for
Nonclassical 21-Hydroxylase Deficiency, J. Clinical Endo. And Metabolism, Vol. 65, No.2, 1987
Otten, B.J., et. Al., Salivary Plasma Androstenedione and 17-Hydroxyprogesterone levels in Congenital
Adrenal Hyperplasia, J. Clin. Endo. And Metab., Vol. 57 No. 5, 1983.
Walker, R.F., et.al., Radioimmunoassay of 17-Hydroxyprogesterone in Saliva, Parotid Fluid, and Plasma ofCongenital Adrenal Hyperplasia Patients, Clin. Chem., 25/4, 542 – 545, 1979.

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Symbols:

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Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContent

CautionCatalogue
numberEffective 2018-06-01

Contains sufficient for<n> tests

For research use
only!
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