HTRF® package insert Human Interleukin 2 ... - Cisbio Bioassays

Headquarters & Europe Office
Cisbio Bioassays
Phone: +33 (0)4 66 79 67 05
Fax: +33 (0)4 66 79 19 20
bioassays@cisbio.com
USA Office
Cisbio US, Inc.
Phone: +1 888 963 4567
Fax: +1 781 687 1500
htrfinfo@cisbio.us
China Office
IBA China
Phone: +86 10 8080 9288
Fax: +86 10 8080 9299
htrfinfo@iba-group.com
Japan Office
Sceti Medical Labo K.K.
Phone: +81 (0)3 5510 2932
Fax: +81 (0)3 5510 0130
reagent@scetimedilabo.co.jp
Human Interleukin 2
20,000 tests
For in vitro research use only
Reagent storage temperature: -60°C or below
www.htrf.com
HTRF ® package insert
Document reference : 64IL2PEC rev 8 (March 2012)
1. Assay description
This assay is intended for the quantitative determination of human IL-2 using the HTRF® technology.
As shown below, IL2 is detected in a sandwich assay format using 2 different specific monoclonal
antibodies, one labelled with Eu 3+ -Cryptate (donor) and the second with d2 (acceptor).
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 (665nm). The two conjugates bind to the antigen present in
the sample, thereby generating FRET. The positive modulation of signal intensity is proportional to
the number of antigen–antibody complexes formed and therefore to the IL2 concentration.
Anti-IL2
d2 conjugate
Anti-IL2
EU 3+ cryptate conjugate
Human IL2
2. Protocol at a glance
Incubate 2h at RT
Read on an HTRF
compatible reader
10µL
Sample or Calibrator
10µL
HTRF conjugates
3. HTRF reagents
Standard
IL2
Anti-(h) IL2-d2-
conjugate
Anti-(h) IL2-
Eu 3+ -Cryptate-conjugate
Diluent
Reconstitution buffer
Green cap Purple cap Red cap White cap Red cap
Stock solution
100µl/vial
[IL2] = 15ng/mL
1ml/vial 1ml/vial 20ml/vial 200ml/vial
Storage -60°C or below -60°C or below -60°C or below 4°C to -60°C* 4°C to -60°C*
Ref #
(when available separately)
N/A N/A N/A 62DL1DDD 62RB3RDF
* Diluent and Reconstitution buffer are shipped frozen, but can be stored at 2-8°C in your premises.

4. Reagent preparation
HTRF® reagent concentrations have been set for optimal assay performances. Note that any dilution or improper use of the d2 and Cryptate-conjugates will impair the assay’s
quality.
For an accurate quantitative determination of sample, dilution must be carried out with the medium used for preparing the samples (i.e. diluent, culture medium or any other
compatible medium).
Standard and conjugates may be frozen and thawed once: to avoid freeze/thaw cycles it is recommended to dispense remaining stock solutions of standard and conjugates into
disposable plastic vials for storage at –60°C or below.
Be careful, working solution preparation may differ between the 1,000 and the 20,000 data point kits.
• Thaw all reagents at room temperature, allow them to warm up (caution: take buffers’ thawing time into account).
• Prepare the working solutions from stock solutions (§3) by following the instructions below.
4.1. Preparation of working solutions of conjugates
Determine the amounts of conjugates needed for the experiment. Each well requires 5µL of each conjugate. In practice:
Anti-(h) IL2-d2 conjugate
Anti-(h) IL2-Eu 3+ -Cryptate- conjugate
1 volume
99 volumes 1 volume
99 volumes
Purple cap
Red cap
Red cap
Red cap
Prepare a 100X diluted solution using the reconstitution buffer: e.g. take 1mL of conjugate stock
solution and add it to 99mL of reconstitution buffer.
Prepare a 100X diluted solution using the reconstitution buffer: e.g. take 1mL of conjugate stock
solution and add it to 99mL of reconstitution buffer.
4.2. Standard curve preparation
Determine how many standard levels and replicates will be tested. Each well requires 10µl of standard.
A recommended standard dilution procedure is listed and illustrated below.
Standards Working concentration (pg/mL) Preparation
Std 8 5000 100µl Calibrator stock solution + 200µl diluent
Std 7 2500 100µl Std 8 + 100µl diluent
Std 6 1250 100µl Std 7 + 100µl diluent
Std 5 625 100µl Std 6 + 100µl diluent
Std 4 312.5 100µl Std 5 + 100µl diluent
Std 3 156.2 100µl Std 4 + 100µl diluent
Std 2 78.1 100µl Std 3 + 100µl diluent
Std 1 39 100µl Std 2 + 100µl diluent
Std 0 0 100µl diluent
→ Dilute the standard stock solution 3-fold with diluent; this yields the high standard (Std 8: 5000 pg/mL) for the top of the curve.
In practice:
• e.g. take 100µL of standard stock solution and add it to 200µL of diluent. Mix gently.
→ Use the high standard (Std 8) to prepare the standard curve using 1/2 serial dilutions as follows:
• Dispense 100µL of diluent in each vial from Std 7 to Std 1.
• Add 100µL of standard 8 to 100µL of diluent, mix gently and repeat the 1/2 serial dilution to make standard solutions: 2500, 1250, 625, 312.5, 156.2, 78.1, 39 pg/mL.
This will create 8 standards for the analyte. Std 0 (Positive control) is diluent alone.
Step 1: Dispense diluent into each vial
200µL 100µL 100µL 100µL 100µL 100µL
100µL
100µL 100µL
Step 2: standards dilution
100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL
Diluent
(white cap)
Std 8 Std 7 Std 6 Std 5 Std 4 Std 3 Std 2 Std 1 Std 0
Standard stock solution
(green cap)
Std 8 Std 7
Std 6 Std 5 Std 4 Std 3 Std 2 Std 1

5. Assay protocol
Dispense the reagents in the following order:
10µL
standard or sample
5µL
anti-IL2-d2 conjugate
5µL
anti-IL2-Eu 3+ cryptate conjugate
The 2 HTRF conjugates can be pre-mix JUST PRIOR to dispensing: DO NOT store the pre-mix solution.
→ Cover the plate with a plate sealer.
→ Incubate at room temperature for 2 hours.
→ Remove the plate sealer and,
→ Read the fluorescence emission at two different wavelengths (665nm and 620nm) on a compatible HTRF® reader.
For more information about HTRF compatible readers, please visit our website at: htrf.com/technology/htrfmeasurement/compatible_readers/
Assay controls
Negative control Cryptate control Buffer control Sample / Std
Used to calculate the
delta F%
Used to check the Cryptate signal
at 620 nm
used to check background
fluorescence
Sample / Std - - - 10µL
Diluent 10µL 10µL 10µL -
Anti-IL2-d2 conjugate 5µL - - 5µL
Anti-IL2-Eu 3+ -Cryptate conjugate 5µL 5µL - 5µL
Reconstitution buffer - 5µL 10µL -
6. Data reduction
This data must not be substituted for that obtained in the laboratory and should be considered only as an example (readouts on PHERAstar plus ). Results may vary from one HTRF
compatible reader to another.
The assay standard curve is drawn up by plotting delta F% versus the analyte concentration:
Standard – pg/mL Ratio (1) CV % (2) Delta F % (3)
Std 0 - Negative control 646 3 0
Std 1 - 39 733 1.5 14
Std 2 - 78.1 766 0.2 19
Std 3 - 156.2 910 2.8 41
Std 4 - 312.5 1 213 1.1 88
Std 5 - 625 1 667 3.2 158
Std 6 - 1250 2 520 2.2 290
Std 7 - 2500 4 345 0.3 573
Std 8 - 5000 7 666 1.8 1087
Delta F %
IL2 standard curve
(Incubation 2H at RT)
1200
1100
1000
900
800
700
600
500
400
300
200
100
0
0 1000 2000 3000 4000 5000 6000
[IL2] pg/mL
Ratio (1)
Signal 665nm
------------------- x 10 4
Signal 620nm
Ratio must be calculated for each individual well.
CV% (2)
Standard deviation
-------------------------- x 100
Mean ratio
The mean and standard deviation can then be worked out from ratio replicates.
DeltaF % (3)
Ratio standard or sample
– Ratio Negative control
---------------------------------------------- x 100
Ratio Negative control
Reflects the signal to background of the assay. The negative control plays the role of an
internal assay control.
For more information about data reduction, please visit our website at: htrf.com/technology/htrfmeasurement/ratio_data_reduction/
To obtain additional information or support, please contact your technical support team (htrfservices@cisbio.com).

7. Assay characteristics
7.1 Calibration
This immunoassay is calibrated against the NIBSC standard IL2 code 86/504. To convert the sample values obtained with HTRF® assay to equivalent NIBSC units, use the equation
below:
NIBSC (86/504) equivalent value (U/ml) = 0.0132 x IL2 value (pg/ml)
7.2 Detection limit
Detection limit (Std 0 + 2 SD): 39 pg/mL
Copyright © 2012 Cisbio Bioassays, France
HTRF®, TRACE®, and the HTRF logo are trademarks belonging to Cisbio Bioassays.
HTRF® products are manufactured under one or more of the following patents and foreign equivalent : EP 0 180 492 / US 4,927,923 / US 5,220,012 / US 5,432,101 – EP 0 321 353 / US 5,457,185 / US 5,534,622 / US 5,346,996 /
US 5,162,508 – EP 0 539 477 / US 5,512,493 – EP 0 539 435 / US 5,627,074 – EP 0 569 496 / US 5,527,684.
Cisbio Bioassays hereby grants to those buying HTRF® products from Cisbio Bioassays or its affiliates / distributors, a worldwide, non-exclusive, royalty-free, limited license to use HTRF® technology with said products for inhouse
life science research only. Signal amplification and correction using HTRF ® technology are covered by the following U.S. patents or patent applications and foreign equivalents : US 5,512,493 – US 5,527,684 – US 6,352,672.