HTRF® package insert Human Interleukin 17A ... - Cisbio Bioassays

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Human Interleukin 17A
20,000 tests
For in vitro research use only
Reagent storage temperature: -20°C or below
www.htrf.com
HTRF ® package insert
Document reference : 64H17PEC rev 10 (August 2012)
1. Assay description
This assay is intended for the quantitative determination of human IL17A using the HTRF® technology.
As on the side, human IL17A 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. Signal
intensity is proportional to the number of antigen–antibody complexes formed and therefore to the
IL17A concentration.
2. Protocol at a glance
Anti-(h)IL17A
d2 conjugate
Anti-(h)IL17A
EU 3+ cryptate conjugate
Human IL17A
Incubate 5h at 18-25°C
Read on an HTRF
compatible reader
10µL
Sample or Calibrator
10µL
HTRF conjugates
(pre-mix)
3. HTRF reagents
(h)IL17A
Standard
Anti-(h)IL17 Ab-d2
conjugate
Anti-(h)IL17A Ab-Eu 3+ -
Cryptate-conjugate
Diluent
Reconstitution buffer
Green cap Purple cap Red cap White cap Red cap
Stock solution
10 µl/vial
300 ng/mL
1 ml/vial 1 ml/vial 20 ml/vial 200 ml/vial
Storage -20°C or below -20°C or below -20°C or below 4°C to -20°C* 4°C to -20°C*
Ref #
(when available separately)
N/A N/A N/A 62DL3DDD 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
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).
Conjugates may be frozen and thawed once: to avoid freeze/thaw cycles it is recommended to dispense remaining stock solutions of conjugates into disposable plastic vials for
storage at –20°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.
• 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 conjugates. In practice:
Anti-(h)IL17A-d2 conjugate
Anti- (h)IL17A-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 50 µL of conjugate
stock solution and add it to 4950 µL of reconstitution buffer
Prepare a 100X diluted solution using the reconstitution buffer: e.g. take 50 µL of conjugate
stock solution and add it to 4950 µL 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 9 5000 200 µl working solution
Std 8 2500 100 µl Std 9 + 100 µl diluent
Std 7 1250 100 µl Std 8 + 100 µl diluent
Std 6 625 100 µl Std 7 + 100 µl diluent
Std 5 312.5 100 µl Std 6 + 100 µl diluent
Std 4 156.25 100 µl Std 5 + 100 µl diluent
Std 3 78.12 100 µl Std 4 + 100 µl diluent
Std 2 39 100 µl Std 3 + 100 µl diluent
Std 1 19.5 100 µl Std 2 + 100 µl diluent
Std 0 0 100 µl diluent
→ Dilute the standard stock solution 60-fold with diluent; this yields the high standard (Std 9: 5000 pg/mL) for the top of the curve.
In practice:
• e.g. take 5µL of standard stock solution and add it to 295 µL of diluent. Mix gently.
→ Use the high standard (Std 9) to prepare the standard curve using 1/2 serial dilutions as follows:
• Dispense 100µL of diluent in each vial from Std 8 to Std 0.
• Add 100µL of standard to 100µL of diluent, mix gently and repeat the 1/2 serial dilution to make standard solutions: 2500, 1250, 625, 312.5, 156.25, 78.12, 39, 19.5 pg/
mL. This will create 9 standards for the analyte. Std 0 (Positive control) is diluent alone.
Step1: Dispense diluent in each vial
100µL 100µL 100µL
295µL
100µL 100µL 100µL 100µL 100µL 100µL
Step 2: Standards dilution
5µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL 100µL
Diluent
(white cap)
Std 9 Std 8 Std 7 Std 6 Std 5 Std 4 Std 3 Std 2 Std 1
Std 0
Standard stock
solution
(green cap)
Std 9
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-(h)IL17A-d2 conjugate
5µL
anti-(h)IL17A-Eu 3+ cryptate conjugate
It is possible to pre-mix the two conjugates just before dispensing and add 10 µl of this mix.
→ Cover the plate with a plate sealer.
→ Incubate at room temperature for 5 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-(h)IL17A-d2 conjugate 5µL - - 5µL
Anti-(h)IL17A-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, after an overnight incubation.
Standard – pg/mL Ratio (1) CV % (2) Delta F % (3)
Std 0 - Negative control 514 2 0
Std 1 - 19.5 543 5 6
Std 2 - 39 592 0 15
Std 3 - 78.1 631 1 23
Std 4 - 156.2 771 0 50
Std 5 - 312.5 1000 2 95
Std 6 - 625 1566 1 205
Std 7 - 1250 2826 2 450
Std 8 - 2500 5344 2 939
Std 9 - 5000 11028 4 2045
DeltaF%
2500
2000
1500
1000
Human IL17A standard curve
Incubation at RT
5h incubation
500
Overnight incubation
0
0 1000 2000 3000 4000 5000 6000
[Human IL17] 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
Detection limit (dose of mean zero + 3SD)
Linear range study
Incubation time
In diluent
28 pg/ml (ON)
19.5 to 5000 pg/ml
5H at RT
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