Instructions for use

17-OH-Progesterone ELISAFR E-2800R

17-OH-Progesterone ELISA
1. INTRODUCTION
1.1 Intended Use
Enzyme immunoassay for the determination of 17-OH-progesterone in human serum and plasma (EDTA).
2. PRINCIPLE
The assay is a solid phase enzyme-linked immunosorbent assay (ELISA) based on the competition principle.
An unknown amount of antigen present in the sample and a fixed amount of enzyme labelled antigen
compete for the binding sites of the antibodies coated onto the wells. After incubation the wells are washedto stop the competition reaction. After the substrate reaction the intensity of the developed colour isinversely proportional to the amount of the antigen in the sample. Results of samples can be determined
directly using the standard curve.
3. WARNINGS AND PRECAUTIONS
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This kit is for research use only.
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.
All human source material used in the preparation of the reagents has been tested and found negative
for antibody to HIV 1&2, HbsAg, and HCV. No test method however can offer complete assurance that
HIV, HBV, HCV or other infectious agents are absent. Therefore, the reagents should be handled in thesame manner as potentially infectious material.
The microplate contains break apart 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 coloured. 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 (18 - 25 °C) before starting the test. Temperature willaffect 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 different
conditions and the binding characteristics of the plates may result slightly different.
Avoid contact with Stop Solution. It may cause skin irritation and burns.
Chemicals and prepared or used reagents have to be treated as hazardous waste according to thenational biohazard safety guideline or regulation.
For information 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
FR E-2831
Content:

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Microtiterplate
12x8 (break apart) strips, 96 wells; Wells coated with an anti-17-OH-progesterone
antibody (polyclonal).

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Standards and Controls - Ready to use
Cat. no.

Component

Concentration

Volume/Vial

FR E-2801

0 ng/ml

1 ml

FR E-2802

0.1 ng/ml

0.5 mlFR E-2803

0.4 ng/ml

0.5 mlFR E-2804

1.6 ng/ml

0.5 mlFR E-2805

6.5 ng/ml

0.5 mlFR E-2806

25 ng/ml

0.5 mlFR E-2851*

For control values and ranges
please refer to QC-Report!

FR E-2852*

0.5 ml
0.5 ml* containing 17-OH Progesterone in serum.
FR E-2840

Enzyme Conjugate - Ready to use

Content:

17-OH-progesterone conjugated to horseradish peroxidase

Volume:1 x 11 ml
Substrate Solution - Ready to use

AR E-0055
Content:

Tetramethylbenzidine (TMB)

Volume:1 x 22 ml

Hazards
identification:
H360D May damage the unborn child.
AR E-0080
Content:

Stop Solution - Ready to use
contains 2 N acidic solution.
Avoid contact with the stop solution. It may cause skin irritations and burns.

Volume:1 x 7 ml

Hazards
identification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.
H335 May cause respiratory irritation.
Wash Solution - 10X concentrated

AR E-0030
Volume:1 x 50 ml

see “Reagent Preparation”
4.2 Material required but not provided

Microcentrifuge
A calibrated microtiter plate reader (450±10 nm)
Microplate mixer operating at about 600 rpm (optional)
Vortex mixer
Calibrated variable precision micropipettes (25 µl, 50 µl 100 µl, 200 µl).
Absorbent paper
Distilled or deionized waterTimer
Semi logarithmic graph paper or software for data reduction

4.3 Storage conditions
When stored at 2 - 8 °C unopened reagents will be stable until expiration date. Do not use reagents beyond
this date. Opened reagents must be stored at 2 - 8 °C. After first opening the reagents are stable for 30days if used and stored properly.
Microtiter wells must be stored at 2 - 8 °C. Once the foil bag has been opened, care should be taken to close
it tightly again.

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4.4 Reagents preparations
Allow the reagents and the required number of wells to reach room temperature (18 - 25 °C) before starting
the test.
Wash Solution
Add deionized water to the 10X concentrated Wash Solution. Dilute 50 ml of concentrated Wash Solution
with 450 ml deionized water to a final volume of 500 ml. The diluted Wash Solution is stable for 12 weeks atroom temperature.
4.5 Disposal of the kits
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 of the test kit or components, the manufacturer has to be informed in writing
within one week after receiving the kit. Severely damaged single components should not be used for a testrun. 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
For determination of 17-OH-Progesterone 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 17-OH-Progesterone concentrations higher
than the highest standard (25 ng/ml) should be diluted with Standard A before assay. The additional
dilution step has to be 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 (18 -25°C) before use. Allreagents 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.

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

2.

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

3.

5.

Dispense 100 µl Enzyme Conjugate into each well.
Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.
Incubate for 60 minutes at room temperature. Shaking on a horizontal shaker during incubation is notnecessary, but it slightly improves the sensitivity of the test.
Briskly empty the contents of the wells by aspiration or by decanting.
Rinse the wells 4 times with diluted Wash Solution (300 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 thewashing procedure!

6.

Add 200 µl of Substrate Solution to each well.

7.

Incubate for 30 minutes in the dark at room temperature.

8.

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

9.

Determine the absorbance of each well at 450±10 nm.
It is recommended that the wells be read within 15 minutes.

4.

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6.3 Calculation of results
1. Calculate the average absorbance values for each set of standards, controls and samples.
2. Construct a standard curve by plotting the mean absorbance obtained from each standard against its
concentration with absorbance value on the vertical (Y) axis and concentration 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 ParameterLogistics) 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 read directly from this standard curve. Samples withconcentrations higher than that of the highest standard have to be further diluted with Standard A. For
the calculation of the concentrations this dilution factor has to be taken into account.
Conversion to SI units:
17-OH-Progesterone (ng/ml) x 3.03 = nmol/l
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

0.0 ng/ml

Standard B 0.1 ng/ml
Standard C 0.4 ng/ml
Standard D 1.6 ng/ml
Standard E 6.5 ng/ml
Standard F 25 ng/ml

OD (450 nm)

3.025
2.653
2.308
1.638
0.904
0.443

7. EXPECTED NORMAL VALUES
Apparently healthy subjects show the following values of 17-OH-Progesterone:
Children

Reproductive aged women

Follicular phase:

0.05 - 2.0 ng/ml
0.1 - 1.0 ng/ml

Luteal phase:

0.6 - 2.5 ng/ml

Ovulation:

0.3 - 1.5 ng/ml

3 -14 years

Post ACTH:
Third trimester:
Postmenopausal women:< 3.2 ng/ml
2.0 - 12 ng/ml
0.13 - 0.6 ng/ml
0.5 - 3 ng/ml

Normal men

It is strongly recommended that each laboratory establishes its own range of normal values.
8. QUALITY CONTROLGood laboratory practice requires to run controls with each standard curve. A statistically significant numberof 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 useof control samples is advised to assure the day to day validity of results. Use controls at both normal andpathological levels. The controls and the corresponding results of the QC-Laboratory are stated in the QCReport added to the kit. The values and ranges stated on the QC sheet always refer to the current kit lot andshould 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 andtrends. 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 andtiming devices, photometer, expiration dates of reagents, storage and incubation conditions, aspiration andwashing methods. After checking the above mentioned items without finding any error contact yourdistributor or the manufacturer directly.

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5/89. PERFORMANCE CHARACTERISTICS
9.1 Analytical Sensitivity
The analytical sensitivity of the 17-OH Progesterone ELISA was calculated by subtracting 2 standard
deviations from the mean of twenty (20) replicate analyses of Standard A. The analytical sensitivity of theassay is 0.0014 ng/ml.
9.2 Specificity (Cross Reactivity)
The following materials have been evaluated for cross reactivity.
Substance% Cross-reactivity

Androstendione< 0.1 %

Testosterone< 0.1 %

Cortisol

0.05 %

11-Desoxycortisol

2.71 %

Cortisone

0.19 %

Corticosterone< 0.1 %

11-Deoxycorticosterone

0.26 %

Progesterone

2.9 %Estradiol< 0.1 %

Estriol< 0.1 %

Estrone< 0.1 %

Pregnenolone

0.17 %

Prednisolone< 0.1 %

Prednisone

0.11 %

DHEA< 0.1 %

DHEA-S< 0.1 %

Danazol< 0.1 %

Dexamethasone< 0.1 %

9.3 Assay Dynamic Range
The range of the assay is between 0.1 - 25 ng/ml.
9.4 Reproducibility
9.4.1 Intra-Assay
The intra-assay variation was determined by 20 replicate measurements of 3 serum samples within one runusing the 17-OH Progesterone ELISA. The intra-assay variability is shown below:
Sample 1

Sample 2

Sample 3

Mean (ng/ml)

0.52

2.29

5.86

SD (ng/ml)

0.05

0.16

0.38

CV (%)

8.9

6.9

6.6

n=202020

9.4.2 Inter-Assay
The inter-assay variation was determined by duplicate measurements of 3 serum samples in 10 different
runs using the 17-OH Progesterone ELISA. The inter-assay variability is shown below:
Sample 1

Sample 2

Sample 3

0.66

2.20

4.91

SD (ng/ml)

0.1

0.18

0.31

CV (%)

14.9

8.0

6.4

n=101010

Mean (ng/ml)

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Recovery was determined by adding increasing amounts of the analyte to three different samples containing
different amounts of endogenous analyte. All samples were then measured by the 17-OH-progesterone
assay procedure. The percentage recoveries were determined by comparing expected and measured valuesof the samples.

SerumSpiking

Measuredconcentration(ng/ml)Expected
concentration(ng/ml)Recovery %12 ng/ml
4 ng/ml
6 ng/ml

0.64
2.55
3.69
4.95

2.64
4.64
6.64

97 %
80 %
75 %

22 ng/ml
4 ng/ml
6 ng/ml

0.66
2.57
4.16
5.47

2.66
4.66
6.66

97 %
89 %
82 %

32 ng/ml
4 ng/ml
6 ng/ml

2.66
5.68
7.39
9.87

4.66
6.66
8.66

122 %
111 %
114 %

9.6 Linearity
Three serum samples containing different amounts of analyte were assayed undiluted and diluted with theStandard matrix (Standard A). The percentage recovery was calculated by comparing the expected andmeasured values for 17-OH-progesterone.

SerumDilution

Measuredconcentration(ng/ml)Expected
concentration(ng/ml)Recovery %1

1 in 2
1 in 41 in 821.06
10.00
4.98
2.49

10.53
5.26
2.6395 %
95 %
95 %

2

1 in 2
1 in 41 in 8

15.06
7.84
4.54
2.33

7.53
3.77
1.88

104 %
120 %
124 %

3native
1 in 2
1 in 41 in 8

23.38
11.43
5.49
2.49

11.69
5.84
2.92

98 %
94 %
85 %

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 Drug Interferences
Until today no substances (drugs) are known to us, which have an influence to the measurement of 17-OHprogesterone in a sample.

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11. REFERENCES
1.

Abraham, G.E., R.S. Swerdloff, D. Tulchinsky et al: Radioimmunoassay of plasma 17hydroxyprogesterone. J. Clin. Endocrinol. Metab. 33:42, 1971
2. Chrousos, G.P., D. L. Loriaux, D.L. Mann, et al: Late onset 21- hydroxylase deficiency mimicking
idiopathic hirsutism or polycystic avarian disease. Annals Intern. Med. 96:143, 1982.
3. Buster, J.E., R.J. Chang, D.L. Preston, et al: Interrelationships of circulating maternal steroids;
progesterone, 16α-hydroxyprogesterone, 17α-hydroxyprogesterone, 20α-dihydroprogesterone, gamma5-pregnolonone, gamma-5- pregnolonone-sulfate, gamma-5-pregnolone-sulfate and 17-hydroxygamma-5-pregnolonone, J. Clin. Endocrinol. Metab. 48:133, 1979.
4. New, M.I., B. Dupont, S. Pang, et al: An update on congenital adrenal hyperplasia. Recent Progress inHormone Research, 37:105, 1981.
5. J. Hotchkiss, A. Drash, et al: Micro filter paper method for 17α-hydroxyprogesterone
radioimmunoassay: Its application for rapid screening for congenital adrenal hyperplasia. J. Clin.
Endocrinol. Metab., 45:1003, 1977.
6. Lobo, R.A., U. Goebelsmann: Adult manifestation of congenital adrenal hyperplasia due to incomplete
21-hydroxylase deficiency mimicking polycystic ovarian disease. Am. J. Obstet. Gynecol., 138:720,
1980.
7. Urban, M.D., P.A. Lee and C.J. Migeon: Adult high infertility in men with congenital adrenalized
hyperplasia. N. Engl. J. Med. 299:1392, 1978.
8. Meikle, A.W., R.J. Worley and C.D. West: Adrenal corticoid hyper-response in hirsute women. Fertil.
Steril. 41:575, 1984
9. Ueshiba, H., Zerah M., New M. I.: Enzyme-linked Immunosorbent assay (ELISA). Method for screeningof non-classical steroid 21-Hydroxylase deficiency. Norm. Metab. Res. 26:43, 1994
10. Liovic M et al. CYP17 gene analysis in hyperandrogenised women with and without exaggerated 17hydroxyprogesterone response to ovarian stimulation. J. Endocrinol. Invest., 20:189, 1997

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