ALLERGEN Total IgE REF - Radim S.p.A.

9.0 ASSAY PROCEDURE
The following steps are required by both the manual and the automated assay procedure on Adaltis and
Radim instruments.
9.1 REAGENTS PREPARATION
• Allow all reagents and the microplate to reach room temperature before use.
• Place the exceeding strips and the desiccant into the transparent zip pouch, seal it properly and
store at 2…8°C.
• Prepare the working wash solution.
9.2 PIPETTING AND INCUBATION STEPS
1. Prepare the assay map reserving the first wells for the calibration curve.
2. Pipette 150 µl of incubation buffer in all the wells.
3. Pipette 25 µl of calibrators and samples into the appointed wells.
4. Shake gently the microplate for 10 sec. being careful not to let the content come out from the
wells.
5. Incubate at 37°C for 60 min., without covering the plate.
6. Wash 3 times (see Section 9.3 Procedural Notes).
7. Dispense 100 µl of anti-IgE-Biotin into all the wells.
8. Incubate at 37°C for 30 min. without covering the plate.
9. Wash 3 times (see Section 9.3 Procedural Notes).
10. Dispense 100 µl of HRP-Streptavidin Conjugate into all the wells.
11. Incubate at 37°C for 30 min. without covering the plate.
12. Wash 3 times (see Section 9.3 Procedural Notes).
13. Dispense 100 µl of Substrate HS into all the wells.
14. Incubate 15 min. at room temperature.
15. Add 100 µl of Stop Solution in the same sequence and timing adopted to dispense the substrate
and read at 450 nm with reference filter set at 620 nm. Read the plate within 60 min.
9.3 PROCEDURAL NOTES
• WASHING PROCEDURE. The use of an automatic plate wash equipment is recommended. After
the washing, tap the inverted plate on absorbent paper to remove any residual from the wells, if
the washer does not aspirate completely the wash solution. On Adaltis and Radim instruments,
once the washing has been correctly programmed, this step is not necessary. Three washings
are required.
• INCUBATION TEMPERATURE. The incubation temperature for all the immunological reaction
must be kept constant at 37°C ± 1°C .
• DISPENSATION OF THE SUBSTRATE HS SOLUTION
In order to obtain precise and accurate results it is necessary to dispense the Substrate HS
immediately after the washing.
It is recommended to time the addition of the Substrate HS and Stop Solution until a good
familiarity with the method is acquired (i.e. if the Substrate HS solution is dispensed into the wells
every 3 seconds one from the other, also the Stop Solution should be dispensed in the same
order and with the same frequency).
The use of repetitive pipettes is particularly convenient.
The contamination of solutions should be avoided. Incubation in the dark is not necessary.
However, avoid a direct exposure to sunlight.
A1002 – ALLERGEN Total IgE
M541 – Rev. 1 – 06/2010 – Pag. 14/32