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

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