Instructions for use

Progesterone ELISAFR E-2500R

Progesterone ELISA
1. Introduction
1.1 Intended Use
The Progesterone ELISA is an enzyme immunoassay for the quantitative measurement of Progesterone inserum or plasma (EDTA, lithium heparin or citrate plasma).
2. Principle of the test
The Progesterone 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 a unique antigenic site of
the Progesterone molecule.
During the first incubation, the progesterone in the added sample competes with the added enzyme
conjugate, which is a progesterone molecule conjugated to horseradish peroxidase, for binding to the coatedantibody.
After a washing step, to remove all unbound substances, the solid phase is incubated with the substratesolution. The colorimetric reaction is abruptly stopped by addition of stop solution and optical density (OD)of the resulting yellow product is measured. The intensity of color is inversely proportional to theconcentration of the analyte in the sample.
A standard curve is constructed by plotting OD values against concentrations of standards, andconcentrations of unknown samples are determined using this standard curve.
3. Warnings and precautions
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For professional 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 (20 °C - 26 °C) before starting the test. Temperature
will affect the optical density readings of the assay. However, values for the 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.
All indicated volumes have to be performed according to the protocol. Optimal test results are only
obtained when using calibrated pipettes and microtiter plate 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 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
Microtiterwells

FR E-2531
Contents:

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

Standards - ready to use
Cat. no.

Component

FR E-2501
FR E-2502
FR E-2503
FR E-2504
FR E-2505
FR E-2506
FR E-2507

Standard

Concentration ng/ml

Volume / vial

Standard A
Standard B
Standard C
Standard D
Standard E
Standard F
Standard G00.31.25
2.551540

1 ml
1 ml
1 ml
1 ml
1 ml
1 ml
1 ml

Contents:

Contain non-mercury preservative.

Conversion:

1 ng/ml = 3.18 nmol/l

FR E-2540

Enzyme Conjugate - ready to use

Content:

Progesterone conjugated to horseradish peroxidase;
Contains non-mercury preservative.

Volume:1 x 25 ml

SA E-0055

Substrate Solution - ready to use

Content:

Tetramethylbenzidine (TMB).

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

FR E-0080
Content:

contains 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 - 40X concentrated

FR E-0030
Volume: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-A calibrated microtiter plate reader (450 nm, with reference wavelength at 620 nm to 630 nm)
Calibrated variable precision micropipettes.
Absorbent paper.
Distilled waterTimer
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.
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.
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4.4 Reagent Preparation
Bring all reagents and required number of strips to reach room temperature (20 °C – 26 °C) 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 thisproduct is given in the Safety Data Sheets, section 13.
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,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 plasma (EDTA, lithium-heparin- or citrate plasma) can be used in this assay.
Note: Samples containing sodium azide should not be used in the assay.
In general it should be avoided to use haemolytic, icteric or lipaemic specimens. For further informationrefer to chapter “Interfering Substances”.
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 7 days at 2 °C – 8 °C prior to assaying.
Specimens held for a longer time (up to 12 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 bediluted 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 Serum + 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-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.
Optical density 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 holder.

2.

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

3.

Incubate for 5 minutes at room temperature.

4.

Dispense 200 µl Enzyme Conjugate into each well.
Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.

5.

Incubate for 60 minutes at room temperature.

6.

Rinse the wells 3 times with 400 µl diluted Wash Solution per well, if a plate washer is used - or Briskly shake out the contents of the wells.
Rinse the wells 3 times with 300 µl diluted Wash Solution per well for manual washing. 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!

7.

Add 200 µl of Substrate Solution to each well.

8.

Incubate for 15 minutes at room temperature.

9.

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

10.

Determine the optical density (OD) of the solution in each well at 450 nm (reading) and at 620 nm
to 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 optical density (OD) values for each set of standards, controls and samples.
2. Using linear graph paper, construct a standard curve by plotting the mean OD obtained from eachstandard against its concentration with OD value on the vertical (Y) axis and concentration on thehorizontal (X) axis.
3. Using the mean OD value for each sample determine the corresponding concentration from the standard
curve.
4. Automated method: The results in the Instructions for Use have been calculated automatically using a 4Parameter 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 > 40
ng/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

Optical Units (450 nm)

Standard A (0 ng/ml)

1.52

Standard B (0.3 ng/ml)

1.17Standard C (1.25 ng/ml)

0.88Standard D (2.5 ng/ml)

0.69

Standard E (5.0 ng/ml)

0.55

Standard F (15 ng/ml)

0.35Standard G (40 ng/ml)

0.13

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7. Expected values
It is strongly recommended that each laboratory should determine its own normal and abnormal values.
In a study conducted with apparently healthy adults, using the Progesterone ELISA the following data wereobserved:nMean(ng/ml)Median(ng/ml)

49

0.36

0.34

0.05 - 0.92

0.05 - 0.94

Follicular Phase

35

0.79

0.76

0.21 - 1.72

0.21 - 1.80

Luteal Phase

45

12.89

13.00

3.78 - 24.60

2.90 - 27.10

Postmenopausal

28

0.53

0.59

0.18 - 0.83

0.15 - 0.84

PopulationMales

2.5th - 97.5th Percentile Range (min. - max.)(ng/ml)(ng/ml)Females

8. Quality Control
Good 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 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 donors 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 0.140 ng/ml – 40.0 ng/ml.
9.2 Specificity of Antibodies (Cross Reactivity)
The following substances were tested for cross reactivity of the assay:
Steroid
Progesterone
17α OH Progesterone
Estriol
Estradiol 17β
Testosterone
11-Desoxycorticosterone
DHEA-S
Cortisol
Corticosterone
Pregnenolone
Cortison
11-Desoxycortisol

Version: 6.0-r

Cross Reaction (%)
100.000.30< 0.10< 0.10< 0.101.10< 0.02< 0.020.20
0.35< 0.100.10

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9.3 Sensitivity
The analytical sensitivity of the Progesterone ELISA was calculated by subtracting 2 standard deviations tothe mean of 20 replicate analyses of the Standard A and was found to be 0.045 ng/ml.
The Limit of Blank (LoB) is 0.120 ng/ml.
The Limit of Detection (LoD) is 0.140 ng/ml.
The Limit of Quantification (LoQ) is 0.144 ng/ml.
9.4 Reproducibility
9.4.1 Intra Assay
The within assay variability is shown below:
SamplenMean (ng/ml)

CV (%)

12
3

20
2020

0.6
4.7
10.8

5.4
7.0
6.9

9.4.2 Inter Assay
The between assay variability is shown below:
SamplenMean (ng/mL)

CV (%)

12
3

121212

0.6
4.6
10.7

10.0
4.3
5.6

9.4.2 Inter-Lot
The inter-assay (between-lots) variation was determined by repeated measurements of samples with 3
different kit lots.
Sample12

n1818

Mean (ng/mL)
1.2
38.7

CV (%)
7.2
3.1

9.5 Recovery
Samples have been spiked by adding progesterone solutions with known concentrations.
The recovery (%) was calculated by multiplying the ratio of measured and expected values with 100.
Sample 1

Sample 2

Sample 3

Concentration (ng/ml)

1.6

4.2

11.0

Average Recovery (%)

101.9

104.1

97.0

from

97.8

96.3

90.9

to112.0

109.0

105.6

Range of Recovery (%)

9.6 Linearity
Samples were measured undiluted and in serial dilutions with Standard A. The recovery (%) was calculated
by multiplying the ratio of expected and measured values with 100.
Sample 1

Sample 2

Sample 3

Concentration (ng/ml)

1.64.2

11.0

Average Recovery (%)

102.5

99.1

106.5

from92.0

87.8

104.7

to111.9

110.3

108.6

Range of Recovery (%)

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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 1.8 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 ofProgesterone in a sample.
10.3 High-Dose-Hook Effect
A High-Dose-Hook Effect is not relevant for competitive assays.
11. References / literature
1. Filicori M, Butler JP, Crowley WF Jr. Neuroendocrine regulation of the corpus luteum in the human.
J Clin Invest. 73:1638 1984.
2. Katt JA, Duncan JA, Herbon L, et al. The frequency of gonadotropin releasing hormone stimulation
determines
thenumberofpituitary
gonadotropin
releasinghormonereceptors.
Endocrinology 1985; 116:2113.
3. Csapo AI, Pulkkinen MO, Wiest WG: Effects of lutectomy and progesterone replacement therapy in earlypregnancy patients. Am J Obstet Gynecol 115:759, 1973.
4. Thomas Labor und Diagnose; TH-Books-Verlags-Gesellschaft; 8. Auflage (2012)

Symbols:

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Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContentCaution

Catalogue
numberEffective: 2020-03-19

Contains sufficient for<n> tests

For research use
only!

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