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