PROGESTERONE KITS

PROTOCOL

Part # 6FPROPEG & 6FPROPEH

Test size#: 500 tests (6FPROPEG), 10,000 tests (6FPROPEH) - assay volume: 20 µL
Revision: 02-Jan.2018
Store at: -20°C or below (6FPROPEG); -20°C or below (6FPROPEH)
For research use only. Not for use in diagnostic procedures.

ASSAY PRINCIPLE

Cisbio Bioassays’ Progesterone assay is only intended for quantitative measurement of Progesterone directly
from cell supernatants or purified solutions, using HTRF ® technology.
Progesterone is detected in a competitive assay format using a specific antibody
labeled with Europium Cryptate (donor) and Progesterone labelled with d2 (acceptor).
The detection principle is based on HTRF® technology. When the dyes are in close proximity, the excitation of
the donor with a light source (laser or flash lamp) triggers a Fluorescence Resonance Energy Transfer (FRET)
towards the acceptor, which in turn fluoresces at a specific wavelength (665 nm). The Progesterone present in
the sample competes with the binding between the two conjugates and thereby prevents FRET from occurring.
The specific signal is inversely proportional to the Progesterone concentration (Fig. 1).

sample standard

Progesterone Progesterone

Anti-Progesterone Progesterone
donor Antibody acceptor Progesterone-acceptor

Figure 1: Principle of HTRF® Progesterone competitive assay.

PROTOCOL AT A GL ANCE

ADD READ ANALYSE

Incubate
1 hour @ RT

[Progesterone] nM
10 µL 5 µL 5 µL
Standard Progesterone Anti-Progesterone
or Sample acceptor donor Antibody

Do not pre-mix the d2 and Cryptate solutions prior

to dispense.

Make sure you use the approriate setup for Eu 3+ Cryptate. For more information about set-up and HTRF® compatible
readers, please visit our website at: http://www.cisbio.com/compatible-readers
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MATERIALS PROVIDED:

500 tests * 10,000 tests *

Kit components
Cat # 6FPROPEG Cat # 6FPROPEH
1 vial - 50µl 1 vial - 50µl
Progesterone Standard
318µM 318µM
Frozen
Ref# 6FCUS000 Ref# 6FCUS000
1 vial - 50µl 1 vial - 1mL
Anti-Progesterone-Eu3+ Cryptate Antibody
Frozen - 50 X Frozen - 50 X
1 vial - 50µl 1 vial - 1mL
Progesterone-d2
Frozen - 50 X Frozen - 50 X
Diluent ** 1 vial 1 vial
ready-to-use 20 mL (62DL3DDD) 20 mL (62DL3DDD)

Detection buffer *** 1 vial 1 vial

ready-to-use 7 mL (6FCUS000) 105 mL (6FCUS000)
Detection Buffer# 3 Detection Buffer# 3

* When used as advised, the two available kit sizes will provide sufficient reagents for 500 and 10,000 tests respectively in 20 µL final.
Assay volumes can be adjusted proportionally to run the assay in 96 or 1536 well microplates.
** The Diluent is an alternative to medium to prepare working standard solutions.
*** The Detection buffer is used to prepare working solutions of acceptor and donor reagents.

PURCHASE SEPAR ATELY:

• HTRF ® 96-well low volume plate Ref# 66PL96001
• 384-well low volume plate (20 µL) *
• HTRF ® -Certified Reader **. Make sure the setup for Eu3+ Cryptate is used.

* For HTRF microplate recommendations, please visit http://www.cisbio.com/drug-discovery/htrf-microplate-recommendations

** For a list of HTRF-compatible readers and set-up recommendations, please visit http://www.cisbio.com/compatible-readers

STOR AGE AND STABILIT Y

Store the kit at -20°C or below. Under proper storage conditions, reagents are stable until the expiry
date indicated on the label.
Diluent and detection buffer are shipped frozen, but can be stored at 2-8°C in your premises.

If lyophilized, once reconstituted, antibodies and standard stock solutions may be frozen. They can
be thawed only once. To avoid freeze/thaw cycles, it is recommended to dispense remaining stock
solutions into disposable plastic vials for storage at -20°C or below .
Reagents Volume of Progesterone standard aliquots should not be under 20 μL.

RE AGENT PREPAR ATION

BEFORE YOU BEGIN:

• It is very important to prepare reagents in the specified buffers. The use of an incorrect diluent may affect reagent stability and
assay results.
• Thaw all reagents at room temperature, allow them to warm up.
• Before use, allow diluent and buffer to warm up at room temperature and homogenize them with a vortex.
• Progesterone standards (for standard curve) must be prepared in diluent or in the same medium as the samples.

TAKE CARE TO PREPARE STOCK AND WORKING SOLUTIONS ACCORDING TO THE DIRECTIONS FOR THE KIT
SIZE YOU HAVE PURCHASED.
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TO PREPARE STANDARD & DETECTION REAGENTS STOCK SOLUTIONS:

500 TESTS KIT - 6FPROPEG 10,000 TESTS KIT - 6FPROPEH

Anti-Progesterone-Eu3+ Cryptate antibody

Thaw the Anti-Progesterone-Eu3+ Cryptate antibody . Thaw the Anti-Progesterone-Eu3+ Cryptate antibody .
Mix gently. This 50 X stock solution can be frozen and Mix gently. This 50 X stock solution can be frozen and
stored at -20°C or below. stored at -20°C or below.

Progesterone-d2
Thaw the Progesterone-d2 . Thaw the Progesterone-d2 .
Mix gently. This 50 X stock solution can be frozen and Mix gently. This 50 X stock solution can be frozen and
stored at -20°C or below. stored at -20°C or below.

Progesterone Standard
Thaw the Progesterone standard solution in order Thaw the Progesterone standard solution in order
to obtain a 318µM (see vial label) stock solution. Mix to obtain a 318µM (see vial label) stock solution. Mix
gently. gently.

TO PREPARE WORKING ANTIBODY SOLUTIONS:

Each well requires 5 µL of Anti-Progesterone-Eu3+ Cryptate Antibody and 5 µL of Progesterone-d2.
Prepare into separate vials the two antibody solutions.

500 TESTS KIT - 6FPROPEG 10,000 TESTS KIT - 6FPROPEH

Anti-Progesterone- Cryptate antibody

Dilute 50-fold the stock solution of anti- 1 vol 49 vol Dilute 50-fold the stock solution of anti-
Progesterone-cryptate antibody with detection 1 vol 49 vol Progesterone-cryptate antibody with detection
buffer#3 e.g. take 0.05 mL of cryptate-antibody stock buffer#3 e.g. take 1 mL of cryptate-antibody stock
solution and add it to 2.45 mL of detection buffer #3. solution and add it to 49 mL of detection buffer #3.

Progesterone-d2
Dilute 50-fold the stock solution of Progesterone-d2 1 vol 49 vol Dilute 50-fold the stock solution of Progesterone-d2
with detection buffer#3: e.g. take 0.05 mL of d2 1 vol 49 vol with detection buffer#3: e.g. take 1 mL of d2 stock
stock solution and add it to 2.45 mL of detection solution and add it to 49 mL of detection buffer #3.
buffer #3.

Antibody mix
Do not pre-mix the d2 and Cryptate solutions prior to Do not pre-mix the d2 and Cryptate solutions prior to
dispense. dispense.
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TO PREPARE WORKING STANDARD SOLUTIONS:
• Each well requires 10 µL of standard.
• D ilute the standard stock solution serially with diluent .
• In order to check for a potential interference effect from your assay buffer when using the assay for the first time, we highly
recommend the parallel preparation of a standard curve in your own supplemented cell culture medium and in diluent.
• In order to counteract any standard sticking, we recommend changing tips between each dilution.
• If the sample to test is a cell supernatant, replace the diluent by culture medium.

A recommended standard dilution procedure is listed and illustrated below:

Dilute the standard stock solution 100-fold with diluent; this yields the Intermediate Standard Solution # A (3,180 nM). e.g: take 5
µL of standard stock solution and add it to 495 µL of diluent. Mix gently.
Dilute the Intermediate Standard#A 47.7-fold with diluent to prepare high standard (Std 8): take 10µl of Intermediate Standard#A
and add it to 467µL of diluent. Mix gently.
Use the high standard (Std 8) to prepare the standard curve using 1/3 serial dilutions as follows:
• Dispense 200µl of diluent into each vial from Std 7 to Std 0.
• Add 100µl of standard to 200µl of diluent, mix gently and repeat the 1/3 serial dilution to make standard solutions:
std7, std6, std5, std4, std3, std2, std1.
This will create 8 standards for the analyte. Std 0 (Positive control) is diluent or appropriate culture medium alone.

5µL 10µl 100µl 100µl 100µl 100µl

Std Std Std Std Std Std

8 7 .. .. 1 0

Progesterone
495µL 467µL 200µl
diluent or appropriate medium diluent or appropriate medium

* Standard curve can be prepared in other appropriate medium

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TO PREPARE SAMPLES:
• Each well requires 10 µL of sample.
• J ust after their collection, put the samples at 4°C and test them immediately. For later use, samples should be dispensed into
disposable plastic vials and stored at -60°C or below. Avoid multiple freeze/thaw cycles
• A
 ll samples with a concentration above the highest standard (Std 8) must be diluted in diluent or in your appropriate sample
medium.

ASSAY PROTOCOL

Negative control (or Cryptate

Standard (Std 0 - Std 8) Sample
control)
Dispense 10 µL of each Progesterone
Dispense 10 µL of diluent into each Dispense 10 µL of sample into each
Step 1

standard (Std 0 - Std 8) into each

diluent well. sample well.
standard well.
Step 2

Add 5 µL of detection buffer to all

Add 5 µL of Progesterone-d2 working solution to all wells
wells
Step 3

Add 5 µL of Anti-Progesterone-Eu3+ Cryptate working solution to all wells

Step 4

Seal the plate and incubate 1 hour @ RT

Step 5

Remove the plate sealer and read on an HTRF® compatible reader

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1 2 3 4 5 6
10 µL Diluent (Negative control) 10 µL Sample 1
A Repeat Well A1 Repeat Well A1 Repeat Well A4 Repeat Well A4
5 µL detection buffer 5 µL Progesterone-d2
5 µL Anti-Progesterone-Eu3+ Cryptate 5 µL Anti-Progesterone-Eu3+ Cryptate
10 µL Std 0 (Positive control) 10 µL Sample 2
B Repeat Well B1 Repeat Well B1 Repeat Well B4 Repeat Well B4
5 µL Progesterone-d2 5 µL Progesterone-d2
5 µL Anti-Progesterone-Eu3+ Cryptate 5 µL Anti-Progesterone-Eu3+ Cryptate
10 µL Std 1 10 µL Sample 3
C Repeat Well C1 Repeat Well C1 Repeat Well C4 Repeat Well C4
5 µL Progesterone-d2 5 µL Progesterone-d2
5 µL Anti-Progesterone-Eu3+ Cryptate 5 µL Anti-Progesterone-Eu3+ Cryptate
10 µL Std ... 10 µL Sample ...
D Repeat Well D1 Repeat Well D1 Repeat Well D4 Repeat Well D4
5 µL Progesterone-d2 5 µL Progesterone-d2
5 µL Anti-Progesterone-Eu3+ Cryptate 5 µL Anti-Progesterone-Eu3+ Cryptate
10 µLStd ... 10 µL Sample ...
E Repeat Well E1 Repeat Well E1 Repeat Well E4 Repeat Well E4
5 µL Progesterone-d2 5 µL Progesterone-d2
5 µL Anti-Progesterone-Eu3+ Cryptate 5 µL Anti-Progesterone-Eu3+ Cryptate
10 µL Std ... 10 µL Sample ...
F Repeat Well F1 Repeat Well F1 Repeat Well F4 Repeat Well F4
5 µL Progesterone-d2 5 µL Progesterone-d2
5 µL Anti-Progesterone-Eu3+ Cryptate 5 µL Anti-Progesterone-Eu3+ Cryptate
10 µL Std ... 10 µL Sample ...
G Repeat Well G1 Repeat Well G1 Repeat Well G4 Repeat Well G4
5 µL Anti-Progesterone-d2 5 µL Progesterone-d2
5 µL Progesterone-Eu3+ Cryptate 5 µL Anti-Progesterone-Eu3+ Cryptate
10 µL Std ... 10 µL Sample ...
H Repeat Well H1 Repeat Well H1 Repeat Well H4 Repeat Well H4
5 µL Progesterone-d2 5 µL Progesterone-d2
5 µL Anti-Progesterone-Eu3+ Cryptate 5 µL Anti-Progesterone-Eu3+ Cryptate
10 µL Std ... 10 µL Sample ...
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
I Repeat Well I1 Repeat Well I1 A Repeat Well I4 Repeat Well I4
5 µL Progesterone-d2 5 µL Progesterone-d2
B
5 µL Anti-Progesterone-Eu3+ Cryptate 5 µL Anti-Progesterone-Eu3+ Cryptate
C
D
E
F
G
E
H
I
J
K
L
M
N
O
P
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DATA REDUCTION & INTERPRETATION

1. Calculate the ratio of the acceptor and donor emission signals for each individual well.
Signal 665 nm
Ratio = x 10 4
Signal 620 nm

2. Calculate the % CVs. The mean and standard deviation can then be worked out from ratio replicates.

Standard deviation
CV (%)= x 100
Mean Ratio

3. Calculate the % delta F which reflects the signal to background of the assay. The negative control plays the role of
an internal assay control. Delta F is used for the comparison of day to day runs of the same assay.

Ratio Standard or sample - Ratio Negative Control

delta F (%) = x 100
Ratio Negative Control

For more information about data reduction, please visit http://www.cisbio.com/htrf-ratio-and-data-reduction

RESULTS

This data must not be substituted for the data obtained in the laboratory and should be considered only as an example (readouts
performed on PHERAstar FS using a 384-low volume white plate). Results may vary from one HTRF® compatible reader to
another.
The assay standard curve is created by plotting delta F% versus the analyte concentration. To determine sample concentration,
we recommend to use a log scale for the Progesterone concentrations and analyze the data with the sigmoidal dose response
curve with variable slope.
This product contains material of biologic origin. Use for research purposes only. Do not use in humans or for diagnostic purposes. The purchaser assumes all risk and responsibility concerning reception, handling and
storage. The use of the cell line will be done with appropriate safety and handling precautions to minimize health and environmental impact. Remaining disclaimer.
Copyright 2016 Cisbio Bioassays. All rights reserved. HTRF and and the HTRF logo are trademarks or registered trademarks of Cisbio Bioassays.

FOR MORE INFORMATION

Europe and other countries +33(0)466-796-705 U.S. and Canada 1-888-963-4567 China +86-21-5018-9880
Japan +81-(0)43-306-8712 Visit www.cisbio.com to find a list of our regional distributors